Tumor therapy with high affinity laminin receptor-targeted vectors

ABSTRACT

The present invention relates to methods and compositions for treating tumors using vectors that preferentially target tumor cells. In particular, the invention relates to alphavirus-based, preferably Sindbis virus-based, vectors and to non-alphavirus-based vectors, which have a preferential affinity for high affinity laminin receptors (HALR). These vectors are efficiently targeted to tumors and have the ability to cause tumor necrosis.

This application is a continuation of Ser. No. 10/473,477, filed Sep.26, 2003, which issued as U.S. Pat. No. 7,306,792 on Dec. 11, 2007,which is a national phase of International Application No.PCT/US02/09432, filed Mar. 27, 2002, which claims priority under 35U.S.C. §119(e) from U.S. Provisional Patent Application Ser. Nos.60/279,051, filed Mar. 27, 2001 and Ser. No. 60/311,373, filed Aug. 10,2001, each of which are incorporated by reference in their entireties.

The research leading to the present invention was supported, in part, byNational Cancer Institute through grant CA 68498. Accordingly, the U.S.government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to viral vector therapies for cancer,particularly to induce apoptosis of tumor cells in a specific manner,relative to normal cells in vivo.

BACKGROUND OF THE INVENTION

Cancer gene therapy would benefit greatly from the availability of avector that has a high efficiency of gene expression and the ability totarget tumors. A number of transfection systems have been developed todeliver heterologous genes into in vivo tumors to investigate cancergene therapy, but all have limitations. For example, retroviral vectorshave been used for gene delivery because they mediate stable genetransfer with a low potential for immunogenicity; however, transferefficiencies are relatively low (see, e.g., Di lanni et al. J.Hematother. Stem. Cell Res., 1999, 8:645-652; Morling et al., GeneTher., 1995, 2:504-508; Lam et al., Hum. Gene Ther., 1996, 7:1415-1422;Kume et al., Stem Cells, 1999, 17:226-232) and germ line modification isa potential problem (see Thompson, Science, 1992, 257: 1854). Inaddition, retroviral vectors, with few exceptions, are susceptible tolysis by serum components in human blood (see Miyao et al., Hum GeneTher, 1997, 8:1575-1583; Russell et al., Hum Gene Ther, 1995, 6:635-641and Rother et al., J Exp Med, 1995, 182:1345-55). This greatly limitstheir in vivo applications. Adenoviral vectors appear to be moreefficient for gene transfer in vivo, but these vectors may be used onlyin a localized manner, because they lack the ability to be delivered viathe bloodstream (see Duncan et al., J Gen Virol., 1978, 40:45-61;Alemany et al., J Gen Virol., 2000, 81 Pt 11:2605-2609 and Alemany etal., Nat. Biotechnol., 2000, 18:723-727), and may cause toxicity topatients due to the highly immunogenic properties of adenoviral proteins(see Ginsberg, Bulletin of the New York Acad. Med., 1996, 73:53-58 andSparer et al., J. Virol., 1997. 71:2277-2284).

Despite these advances, cancer continues to be a major public healthproblem requiring new solutions. Current efforts at developingtherapeutic vectors founder on problems of vector safety and expressionefficacy of the therapeutic gene.

Many properties of alphavirus vectors make them a desirable alternativeto other virus-derived gene delivery systems being developed, includingthe ability to (i) rapidly engineer expression constructs, (ii) producehigh-titered stocks of infectious particles, (iii) infect non-dividingcells, and (iv) attain high levels of expression (Strauss and Strauss,Microbiol. Rev. 1994, 58:491-562; Liljeström et al., Biotechnology 1991,9:1356-1361; Bredenbeek et al., Semin. Virol. 1992, 3:297-310; Xiong etal., Science 1993, 243:1188-1191). Defective Sindbis viral vectors havebeen used to protect mammals from protozoan parasites, helminthparasites, ectoparasites, fungi, bacteria, and viruses (PCT PublicationNo. WO 94/17813).

A cDNA encoding Venezuelan Equine Encephalitis (VEE) and methods ofpreparing attenuated Togaviruses have been described (U.S. Pat. No.5,185,440). Infectious Sindbis virus vectors have been prepared withheterologous sequences inserted into the structural region of the genome(U.S. Pat. No. 5,217,879). In addition, RNA vectors based on the SindbisDefective Interfering (DI) particles with heterologous sequences havealso been described (U.S. Pat. No. 5,091,309). Alphaviruses,specifically the Semliki Forest Virus, were used medically to deliverexogenous RNA encoding heterologous genes, e.g., an antigenic epitope ordeterminant (PCT Publications No. WO 95/27069 and WO 95/07994). Vectorsfor enhanced expression of heterologous sequences downstream from analphavirus base sequence have been also disclosed (PCT Publication No.WO 95/31565). Alphavirus-based vectors were also used for proteinproduction or expression of protein sequences for immunization (PCTPublication No. WO 92/10578). A cDNA construct for alphavirus vectorsmay be introduced and transcribed in animal or human cells (PCTPublication No. WO 95/27044).

Sindbis virus is a member of the alphavirus genus and has been studiedextensively since its discovery in various parts of the world beginningin 1953 (see Taylor et al., Egypt. Med. Assoc., 1953, 36:489-494; Tayloret al., Am. J. Trop. Med. Hyg, 1955, 4:844-862; and Shah et al., Ind. J.Med. Res, 1960, 48:300-308). Like many other alphaviruses, Sindbis virusis transmitted to vertebrate hosts from mosquitos. Alphavirus virionsconsist of a nucleocapsid, wrapped inside a lipid bilayer, upon whichthe envelope proteins are displayed. The envelope proteins mediatebinding to host cell receptors, leading to the endocytosis of thevirion. Upon endocytosis, the nucleocapsid, a complex of the capsidprotein and the genomic viral RNA, is deposited into the cytoplasm ofthe host cell. The Sindbis virus genome is a single-stranded 49S RNA of11703 nt (Strauss et al., 1984, Virology, 133: 92-110), capped at the 5′terminus and polyadenylated at the 3′ terminus. The genomic RNA is of(+)-sense, is infectious, and serves as mRNA in the infected cell.Translation of the genomic RNA gives rise to the nonstructural proteins,nsP1, nsP2, nsP3, and nsP4, which are produced as polyproteins and areproteolytically processed. Early during infection, the nonstructuralproteins, perhaps in association with host factors, use the genomic(+)-sense RNA as template to make a full-length, complementary (−)strand RNA. The (−) strand is template for synthesis of full-lengthgenomic RNA. An internal promoter on the (−) strand is used fortranscription of a subgenomic 26S mRNA which is co-linear with the 3′terminal one-third of the genomic RNA. This 26S subgenomic mRNA istranslated to produce a structural polyprotein that undergoesco-translational and post-translational cleavages to produce thestructural proteins: C (capsid), E2, and E1 (envelope). The capsidprotein C encapsidates the genomic RNA to form nucleocapsids. Theseinteract with the cytoplasmic domain of the cell surface-bound viralenvelope proteins, resulting in the envelopment of the nucleocapsidinside a membrane bilayer containing the envelope proteins, and thebudding of progeny virions out of the infected cell. Sindbis virusinfection has been shown to induce apoptosis in a host cell (Levine etal., Nature, 1993, 361; 739-742; Jan and Griffin, J. Virol., 1999,73:10296-10302).

Although gene transduction based on Sindbis virus has been well-studiedin vitro (see Straus et al., Microbiol. Rev., 1994, 58: 491-562;Altman-Hamamdzic et al., Gene Ther., 1997, 4; 815-822; Gwag et al.,Mole. Brain Research., 1998, 63: 53-61; Bredenbeek et al., J Virol,1993, 67; 6439-6446; Liljestrom et al., Biotechnology, 1991, 9:1356-1361; Piper et al., Meth. Cell Biol., 1994, 43:55-78; and Grusby etal., Proc Natl. Acad. Sci. USA., 1993, 90:3913-3917) and there areseveral reports of in vivo Sindbis virus gene transfer to the centralnervous system (Duncan et al., J Gen Virol., 1978, 40:45-61; Alemany etal., J Gen Virol., 2000, 81 Pt 11:2605-2609; and Alemany et al., Nat.Biotechnol., 2000, 18:723-727) as well as to antigen presenting cells(see Tsuji et al., J Virol, 1998, 72:6907-6910; Hariharan et al., JVirol, 1998, 72:950-958; Pugachev et al., Virology, 1995, 212:587-594;and Xiong et al., Science, 1989, 243: 1188-1191), in vivo use of Sindbisvirus system has been rather limited.

However, a major drawback to the use of Sindbis virus-based vectors wasthe fact that these vectors were thought (prior to this invention) tolack useful target cell specificity. For mammalian cells, at least oneSindbis virus receptor is a protein previously identified as the highaffinity laminin receptor (HALR), whose wide distribution and highlyconserved nature may be in part responsible for the broad host range ofthe virus (Strauss and Strauss, 1994, supra; Wang et al., J. Virol.1992, 66:4992-5001). It was therefore thought desirable to alter thetropism of the Sindbis virus vectors to permit gene deliveryspecifically to certain target cell types (see PCT Publication No. WO98/44132). Such alteration of tropism was suggested to require both theablation of endogenous viral tropism and the introduction of noveltropism, e.g., by engineering a chimeric viral envelope proteincontaining an IgG binding domain of protein A.

In the mature Sindbis virus virion, a (+)-sense viral genomic RNA iscomplexed with capsid protein C to form icosahedral nucleocapsid that issurrounded by lipid bilayer in which two integral membraneglycoproteins, E1 and E2 are embedded (Strauss and Strauss, 1994,supra). Although E1 and E2 form a heterodimer that functions as a unit,the E2 domain appears to be particularly important for binding to cells.Monoclonal antibodies (mAbs) capable of neutralizing virus infectivityare usually E2 specific, and mutations in E2, rather than E1, are moreoften associated with altered host range and virulence (Stanley et al.,J. Virol. 1985, 56:110-119; Olmsted et al., Virology 1986, 148:245-254;Polo et al., J. Virol. 1988, 62:2124:2133; Lustig et al., J. Virol.,1988, 62:2329-2336). Also, a Sindbis virus mutant was identified whichcontained an insertion in E2 and exhibited defective binding tomammalian cells (Dubuisson et al., J. Virol. 1993, 67:3363-3374).

In summary, there remains a need in the art for an effective treatmentfor cancer. In particular, the art needs an effective therapy thatspecifically targets tumor cells for destruction without significantadverse consequences for normal cells. The present invention addressesthese and other needs in the art.

SUMMARY OF THE INVENTION

The present invention provides a novel method for treating a mammal(e.g., human) suffering from a tumor that expresses greater levels ofhigh affinity laminin receptor (HALR) compared to normal cells of thesame lineage. The method of the invention comprises administering to amammal harboring such a tumor an amount of a vector effective to treatthe tumor, wherein the vector has a preferential affinity for HALR.Preferably, the vector is a virus-based vector. As disclosed herein, thepreferred virus-based vector for use in the method of the invention isan alphavirus-based vector (e.g., a replication defectivealphavirus-based vector), more preferably, a replication defectiveSindbis virus-based vector.

As disclosed herein, in addition to alphavirus-based vectors whichpossess natural affinity for HALR and natural apoptosis-inducingfunctions, the vectors of the invention can be derived from any particleor virus that can be effectively modified to have a preferentialaffinity for HALR and to possess an anti-tumor activity. Accordingly, ina separate embodiment, the instant invention includes a method fortreating a mammal suffering from a tumor using the vector which has apreferential affinity for HALR and encodes an anti-tumor gene. Asdisclosed herein, such anti-tumor gene can be a suicide gene, anapoptosis-inducing gene, a tumor suppressor gene, an oncogene antagonistgene, an immunostimulatory gene, a tumor suppressor effector gene, anantisense oligonucleotide-encoding sequence, a ribozyme-encodingsequence, or an immunogenic peptide-encoding sequence. In a preferredembodiment, such anti-tumor gene is an apoptosis-inducing gene or acytokine-encoding gene.

In another embodiment, the present invention provides a method fortreating a mammal (e.g., human) suffering from a tumor using analphavirus-based vector, wherein the vector is not modified tospecifically target the tumor. Preferably, the alphavirus-based vectoris a Sindbis virus-based vector, most preferably, a replicationdefective Sindbis virus-based vector. As disclosed herein, thealphavirus-based vector can be modified to encode a heterologousanti-tumor gene.

The instant invention also includes a method for treating a mammalsuffering from a tumor using the alphavirus-based vector which ismodified to target a specific tumor. Thus, as disclosed herein, thevector for use in the method of the invention can be modified to encodea molecule which specifically interacts with a ligand present in tumorcells. According to a specific embodiment, the vector can be modified toencode, for example, a chimeric envelope protein containing atumor-specific receptor-binding sequence, a peptide mimetic withaffinity for a tumor-specific binding site, an immunoglobilin moleculeor its fragment recognizing a tumor-specific antigen, or the IgG-bindingdomain of protein A that can be administered together with anti-viralreceptor (e.g., anti-HALR) antibodies.

As specified in the Detailed Description and Examples, the methodsaccording to the present invention can be used to treat all kinds oftumors and metastases. In a specific embodiment, the method according tothe present invention is used to treat solid tumors, in particular,hepatic carcinoma, melanoma, epidermoid carcinoma, pancreatic cancer,brain malignancies (such as neuroblastoma, glioblastoma, glioma,medulloblastoma, astrocytoma, acoustic neuroma, oligodendroglioma, andmeningioma), breast cancer, lung cancer (such as small cell lung andnon-small cell lung cancer), ovarian adenocarcinoma, colon cancer,prostate cancer, bladder cancer, and renal cancer.

According to the present invention, the anti-tumor vector is preferablyadministered to the mammal (e.g., human) parenterally, e.g.,intraperitoneally.

The invention further provides the evidence that the disclosed methodsof treating tumors, especially with alphaviral vectors, are particularlyefficient if the mammal undergoing treatment has at least a partiallyfunctional immune system. In a specific embodiment, the inventionprovides the evidence that the methods of treating tumors using Sindbisvirus-based vectors are more efficient in the presence of functionalnatural killer (NK) cells.

Further provided herein is a method for killing a tumor cell (either invivo or in vitro) comprising contacting the tumor cell with an amount ofa vector effective to kill the tumor cell, wherein the vector has apreferential affinity for HALR and may or may not encode a heterologousanti-tumor gene. Preferably, the vector is a replication defectivealphavirus-based vector, more preferably a replication defective Sindbisvirus-based vector.

In conjunction with the methods disclosed herein, the present inventionadvantageously provides pharmaceutical compositions for treating amammal suffering from a tumor comprising a vector and a pharmaceuticallyacceptable carrier or diluent, wherein the vector has a preferentialaffinity for HALR and is effective to kill a tumor, with the provisothat, if the vector is an alphavirus-based vector, it has not beenmodified to target a tumor-specific cellular determinant. Thesepharmaceutical compositions can be used to treat all kinds of tumors andmetastases that are characterized by elevated levels of HALR expressioncompared to normal cells of the same lineage. As disclosed herein, thevectors of the present invention can be derived from particles orviruses that have no natural affinity for HALR (e.g., retroviruses oradenoviruses) by modifying their targeting molecules to include theHALR-binding domain(s) of the Sindbis virus envelope protein(s).Alternatively, the vector can be an alphavirus-based vector, mostpreferably a replication defective Sindbis virus-based vector, which hasits natural targeting properties and has not been modified to target atumor-specific cellular determinant.

The vectors for use in the pharmaceutical compositions of the inventioncan be modified to include a heterologous anti-tumor gene such as butnot limited to a suicide gene, an apoptosis-inducing gene, a tumorsuppressor gene, an oncogene antagonist gene, an immuno-stimulatorygene, a tumor suppressor effector gene, an antisenseoligonucleotide-encoding sequence, a ribozyme-encoding sequence, or animmunogenic peptide-encoding sequence. In a preferred embodiment, suchanti-tumor gene is an apoptosis-inducing gene or a cytokine-encodinggene.

The compositions of the invention can be used to treat various solidtumors, in particular, hepatic carcinoma, melanoma, epidermoidcarcinoma, pancreatic cancer, brain malignancies (such as neuroblastoma,glioblastoma, glioma, medulloblastoma, astrocytoma, acoustic neuroma,oligodendroglioma, and meningioma), breast cancer, lung cancer (such assmall cell lung and non-small cell lung cancer), ovarian adenocarcinoma,colon cancer, prostate cancer, bladder cancer, and renal cancer.

As disclosed in a separate embodiment, the compositions of theinvention, especially the compositions comprising alphaviral vectors,are particularly efficient if administered to mammals (e.g., via aparenteral route) having at least a partially functional immune system,preferably the immune system comprising functional natural killer (NK)cells.

The present invention meets these and other objects of the invention, asset forth in greater detail in the Detailed Description and Examples,including the accompanying Drawings.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of the Sindbis virus-based expressionand helper vectors. SinRep/LacZ is a Sindbis virus-based expressionvector, which contains the packaging signal, nonstructural protein genesfor replicating the RNA transcript and the LacZ gene. DH-BB is aparental helper plasmid that contains the genes for the structuralproteins (capsid, E3, E2, 6K and E1) required for packaging of theSindbis viral genome. Abbreviations: PSG, Sindbis virus subgenomicpromoter; C, capsid; nsp 1-4, nonstructural protein genes 1-4; poly A,polyadenylation signal. Not shown are SinRep/Luc and SinRep/IL12vectors, which are generally similar to SinRep/LacZ. SinRep/Luccomprises a DNA fragment encoding the firefly luciferase gene (Luc) inplace of LacZ (subcloned into the Xba I site of SinRep vector).SinRep/IL12 comprises murine IL12 α-subunit and β-subunit genes(subcloned into the Mlu I site and the Stu I site downstream of the SphI site) and a second subgenomic promoter DNA downstream of the originalsubgenomic promoter of SinRep/LacZ.

DETAILED DESCRIPTION OF THE INVENTION

The present invention advantageously harnesses simple alphavirus vectorsand existing anti-tumor vectors for effective anti-tumor therapy. Theinvention is based, in part, on the unexpected discovery that areplication defective alphavirus vector is an effective anti-cancertherapeutic. Accordingly, the invention provides a method for treating amammal (e.g., human) suffering from a tumor using an alphavirus-basedvector, wherein the vector is not modified to specifically target thetumor. Preferably, the alphavirus-based vector is a Sindbis virus-basedvector, most preferably, a replication defective Sindbis virus-basedvector.

Specifically, the present invention is based, in part, on theobservation that a replication defective Sindbis virus vector, in whichthe structural genes are deleted and substituted with, e.g., aheterologous reporter gene (such as β-galactosidase or luciferase)operatively associated with a Sindbis virus subgenomic promoter proximalto the 5′ end of the coding sequence, and a polyadenylation signal atthe 3′ end of the coding sequence, is able to effectively target tumorcells in vivo. It has been unexpectedly discovered that Sindbis vectors,unmodified with respect to targeting, display high affinity for tumorcells growing in vitro or in vivo and are capable of efficientlyinducing their death leading to tumor regression and long term survivalof experimental tumor-bearing animals (even in the absence of anyheterologous anti-tumor gene payload).

In this respect, the present invention provides an unexpected andadvantageous departure from standard gene therapy using viral vectors.In gene therapy, the vector is a vehicle for delivering a therapeuticgene. In the case of cancer gene therapy, the therapeutic gene adverselyaffects tumor cells. Examples of typical therapeutic genes for genetherapy of cancer (“tumor therapeutic genes”) include, but are notlimited to, tumor suppressors (e.g., p53 and RB); anti-oncogenicintracellular antibodies (e.g., anti-Ras and anti-Raf antibodies); aprotein capable of enzymatically converting a prodrug into a compoundthat is toxic to the tumor (e.g., herpes simplex virus thymidine kinase[HSV-tk], which forms a toxin with ganciclovir; varicella zoster virusthymidine kinase [VZV-tk], which forms a toxin with 6-methoxypurinearabinoside; or a bacterial cytosine deaminase, which forms a toxin with5-fluorocytosine); or an immuno-stimulatory protein capable of enhancinganti-tumor immunity (e.g., flt-3 ligand, GM-CSF, IL-2, IL-7, IL-12, andIL-13). Although the presence of any such “tumor therapeutic gene” inthe vectors of the invention can enhance the anti-tumor effect (asshown, e.g., by comparing a vector encoding a marker gene [LacZ] and avector encoding a cytokine gene [IL12]; see Example 1, infra), it isdisclosed herein that the presence of such gene is not necessary toobtain the therapeutic effect if the vector is an alphavirus vector thatis naturally apoptotic. Accordingly, the Sindbis virus-based vectors ofthe invention need not carry any heterologous coding sequence at all. Ina specific embodiment, however, the therapeutically active Sindbis virusvectors of the invention encode a heterologous marker gene, such asβ-galactosidase, luciferase, or Hygromycin-EGFP. In another embodiment,these vectors encode a heterologous anti-tumor therapeutic gene, such asan apoptosis-inducing gene or a cytokine-encoding gene.

The instant invention takes advantage, for the first time, of thenatural affinity of an alphavirus, particularly Sindbis virus, for tumorcells, in particular, for tumor cells that express higher levels of highaffinity laminin receptors (HALRs), as compared to normal cells of thesame lineage. The term “high affinity laminin receptor” or “HALR” hasits ordinary meaning in the art, i.e., the Mr 67,000 laminin receptorthat can function as the receptor for Sindbis virus entry into cells(see Wang et al., J. Virol. 1992, 66:4992-5001; Strauss et al., Arch.Virol. Suppl. 1994, 9:473-84). Based on this observation, it is clearthat modifying any vector to target it to HALR is within the scope ofthe invention.

Accordingly, the present invention provides a method for treating amammal (e.g., human) suffering from a tumor that expresses greaterlevels of high affinity laminin receptor (HALR) compared to normal cellsof the same lineage. The method comprises administering to a mammalharboring such a tumor an amount of a vector effective to treat thetumor, wherein the vector has a preferential affinity for HALR. Thevector can be derived from any particle or virus that can be effectivelymodified to have a preferential affinity for HALR and to possess ananti-tumor activity.

While not bound by any particular theory, three sets of observations,which have not been previously considered together, may account for theremarkable anti-tumor efficiency of Sindbis vector-based therapy of thepresent invention. First, the HALR can function as the receptor forSindbis virus entry into cells of most species (Wang et al., J. Virol.,1992, 66:4992-5001; and Strauss et al., Arch. Virol. Suppl., 1994,9:473-484). Second, it is widely recognized that expression of the HALR(Mr 67,000) is markedly elevated in many types of cancers (Menard etal., Breast Cancer Res. Treat, 1998, 52:137-145). In fact, a significantcorrelation has been established between the increased expression of Mr67,000 HALR and cancers of the breast (Menard et al., 1998, supra; PaoloViacava et al., J. Pathol., 1997, 182: 36-44; Martignone et al., J.Natl. Cancer Inst., 1993, 85:398-402), thyroid (Basolo et al., Clin.Cancer Res., 1996, 2:1777-1780), colon (Sanjuan et al., J Pathol., 1996,179:376-380), prostate (Menard S et al., Breast Cancer Res. Treat, 1998,52:137-145), stomach (de Manzoni et al., Jpn J Clin. Oncol., 1998,28:534-537), pancreas (Pelosi et al., J Pathol., 1997, 183:62-69), ovary(Menard et al., Breast Cancer Res. Treat, 1998, 52:137-145; and van denBrule et al., Eur J Cancer, 1996, 32A:1598-1602.), melanocytes(Taraboletti et al., J Natl. Cancer Inst., 1993, 85:235-240), lung(Menard et al., Breast Cancer Res. Treat, 1998, 52:137-145), liver(Ozaki et al., Gut, 1998, 43:837-842), endometrium, and uterus (van denBrule et al., Hum Pathol, 1996, 27:1185-1191). Indeed, data on more than4000 cases of different tumors from diverse organs studied byimmunohistochemistry are all concordant with a role for HALR ininvasiveness, metastasis, and tumor growth (Menard et al., Breast CancerRes. Treat., 1998, 52:137-145). Sindbis vectors, which are naturallyblood-borne, can easily travel through the circulation and specificallyhome to growing and metastatic tumors expressing increased levels ofHALR. Finally, Sindbis virus is well known to be highly apoptotic formammalian cells (Levine et al., Nature 1993, 739-742; Jan et al. JVirol., 1999; 10296-10302; Jan et al. J Virol., 2000 6425-6432). Celldeath begins within a few hours of infection and by 48-96 hoursvirtually all infected cells are dead (Sawai et al., Mol Genet Metab.1999, 67:36-42; Griffin et al., Ann. Rev., 1997, Microbiol. 51:565-592).

Although the evidence of HALR involvement in Sindbis virus-mediatedtumor cell infection is compelling, it is possible that Sindbisvirus-based vectors as well as other alphavirus-based vectors of theinvention also interact with other tumor-specific cellular determinants(e.g., receptors). In other words, while the disclosed vectorspreferentially target to tumor cells that express increased levels ofHALRs, this may reflect a characteristic of the cell and need not be themechanism by which the vector infects the cell. The present inventionencompasses any other such mechanisms involved in alphavirus-mediatedinfection and cytotoxicity of tumor cells.

As noted above, vectors of the invention can also carry payloads of oneor several genes, which could be used to enhance the cytotoxicpotential, if necessary. Thus, the invention further relates to usingvectors for delivery of anti-tumor therapeutic genes.

Importantly, compared to tumor cells, vectors of the present invention,especially Sindbis vectors, do not appear to infect normal cells to thesame extent in vivo. This allows for a differential effect in vectortherapy, e.g., where infection by Sindbis vector with resulting death oftumor cells can lead to tumor elimination without apparent deleteriouseffects to other tissues and organs of the treated subjects. Thisphenomenon may be explained by the observation that an increased numberof HALRs in tumors versus normal cells leads to a high number of exposedor unoccupied receptors on tumor cells (Liotta, L. A. Cancer Research,1986, 46:1-7; Aznavoorian et al., 1992, Molecular Aspects of Tumor CellInvasion and Metastasis, pp. 1368-1383). For example, it has beendemonstrated that breast carcinoma and colon carcinoma tissues contain ahigher number of exposed (unoccupied) HALR receptors compared to benignlesions (Liotta et al., 1985, Exp. Cell Res., 156:117-26; Barsky et al.,Breast Cancer Res. Treat., 1984, 4:181-188; Terranova et al., Proc.Natl. Acad. Sci. USA, 1983, 80: 444-448). These excess unoccupied HALRreceptors on tumor cells, which are not found in normal cells, may beavailable for Sindbis virus binding, infection, and induction of celldeath.

Recognition that replication defective Sindbis virus vectors withunmodified cell specificity have intrinsic anti-tumor activity allows totake advantage of the numerous other important properties of Sindbisvectors. Thus, Sindbis vectors show extremely high efficiency of genetransfer. They are (+)-stranded RNA viruses, which through a process ofamplification in the cytoplasm of infected cells can express 105 or moreactive RNA species per cell within a few hours after infection. Thislevel of RNA amplification also allows for very high levels ofexpression of the transferred gene products, which would, in turn, leadto prolonged expression where it not for the apoptotic nature of thevirus (Levine et al., 1993, Nature, 361: 739-742; Jan et al., J Virol.,1999, 73: 10296-10302; Jan et al., J Virol., 2000, 74: 6425-6432;Balachandran et al., J. Virol., 2000, 74: 1513-1523). Based onrecommendations for the handling of alphaviruses and other arbovirusesin the laboratory (The Subcommittee on Arbovirus Laboratory Safety ofthe American Committee on Arthropod-Borne Viruses, Am. J. Trop. Med.Hyg, 1980, 29:1359-1381), Sindbis is considered fairly safe. Themajority of alphaviruses require Level 3 practice and containment and/orvaccination, whereas Sindbis requires only Level 2 practices andcontainment, which is assigned to viruses whose infection result eitherin no disease or in disease which is self-limited. Replication defectiveSindbis vectors derived from Sindbis viruses, as exemplified herein, canbe considered even safer as their capacity to infect and replicate tocause viremia or disease is virtually non-existent. The capacity toinfect and replicate to cause viremia can only be reacquired throughrecombination, which can be minimized and monitored. Sindbis vectorsalso avoid potential complications associated with chromosomalintegration (Xiong et al., Science, 1989, 243: 1188-1191). Recentmethods have added substantial ease to engineering new Sindbis vectorconstructs capable of nonreplicative infection and further enhancedsafety aspects of the vector (Straus et al., Microbiol. Rev., 1994, 58:491-562, 1994). Because Sindbis virus is a blood-borne virus (Turrell,1988, CRC Press, Inc. Boca Raton, Fla.) and can cross the blood-brainbarrier (Altman-Hamamdzic et al., Gene Ther., 1997, 4; 815-822), vectorsbased on this virus are among the few available ones that are capable ofmigrating through the blood stream to reach all cells of the body. Inthis respect they hold an important advantage over many other vectorsand can be used, for example, to treat brain malignancies (such asneuroblastoma, glioblastoma, glioma, medulloblastoma, astrocytoma,acoustic neuroma, oligodendroglioma, and meningioma).

In addition to “naturally targeted” alphavirus-based vectors, thepresent invention also discloses tumor necrosis-inducingalphavirus-based vectors, which carry more specific tumor targetingmolecules, such as, for example, chimeric envelope proteins containing atumor-specific receptor-binding sequence, a peptide mimetic withaffinity for a tumor-specific binding site, an immunoglobulinmolecule/fragment recognizing a tumor-specific antigen, or theIgG-binding domain of protein A that can be administered together withanti-viral receptor (e.g., anti-HALR) antibodies. In a specificembodiment, the present invention provides Sindbis-based vectors, whichare specifically targeted to tumor cells due to the presence of chimericE2 envelope proteins modified, e.g., to include one or more of the fivehighly homologous 58 amino acid-long Fc IgG-binding domains of proteinA, i.e., domains E, D, A, B, C, or domain Z, an engineered analog of theB domain containing two amino acid substitutions Ala1->Val andGly29->Ala (see commonly owned PCT Publication No. WO 98/44132; Uhlen etal., J. Biol. Chem., 1984, 259:1695; Moks et al., Eur. J. Biochem.,1986, 156:637-43). In a further embodiment, the invention providesSindbis virus-based vectors comprising chimeric envelope proteins, whichare modified to include a domain capable of binding to other kinds ofdeterminants expressed at high levels on the surface of a target tumorcell (e.g., EGF receptors overexpressed in many cancer cells or α_(v)β₃integrins overexpressed on melanoma cells; see Dmitriev et al., J.Virol., 2000, 6875-84; Bonnie et al., Virol., 2000, 269:7-17) or,alternatively, a domain interacting with receptors expressed at arelatively higher level in a specific cancer cell type (e.g., ductalepithelial cells in breast cancer). Thus, in a particular embodiment,the present invention provides Sindbis virus-based vectors comprisingchimeric E2 envelope proteins modified by insertion of the α- and β-hCGsequences (as disclosed by the present inventors in Sawai and Meruelo,1998, Biochem. Biophys. Res. Com., 248:315-323) and having the abilityto selectively infect and transfer a reporter gene to choriocarcinomacells as well as other tumor cells bearing LH/CG receptors, but not tocells lacking these receptors.

In contrast to many other viral vectors comprising chimeric targetingmolecules, the alphavirus-based vectors of the present inventioncomprising chimeric E2 proteins are highly tolerant to the specificstructure and/or size of a heterologous targeting fragment inserted inthe E2 envelope protein. This property is likely to be attributed to theseparation of the functional roles between E2 and E1 proteins in celltargeting and fusion. Specifically, extensive experimentation hasestablished that E2 protein is involved primarily in binding of thevirus to the cell surface receptors, while viral entry involves lowpH-induced exposure of the fusion domain in E1 followed by fusion withthe endosomal membrane, endocytosis in clathrin-coated vesicles, andtransfer to endosomes (Hoekstra et al., Biosci Rep., 1989, 9:273-305;Kielian and Helnius, pp. 91-119, In S. Schlesinger and M. J. Schlesinger(ed.), The Togaviridae and Flaviviridae. Plenum Publishing Corp., NewYork, 1986; Kielian et al., J. Virol., 1990, 64:4614-24; Marsh, BiochemJ., 1984, 218:1-10; Stegmann et al., Annu. Rev. Biophys. Biophys. Chem.,1989, 18:187-211; Helenius et al., J. Cell Biol., 1980, 84:404-20; Marshet al., Cell, 1983, 32: 931-940).

Tumor-specific targets for the chimeric envelope proteins of the vectorsof the instant invention include without limitation any tumorcell-specific protein, peptide, oligonucleotide, lipid, polysaccharide,and a small molecule ligand.

Most importantly, the present invention is not limited to naturallytargeted alphavirus-derived anti-tumor vectors. As specified herein,using methods that are well known in the art, the vectors of the presentinvention can be derived from viruses (such as retroviruses,adenoviruses, adeno-associated viruses, lentiviruses, herpes viruses,vaccinia viruses, baculoviruses, papilloma viruses, etc.) or non-viralparticles (such as liposomes, microspheres, protein matrices, etc.) thathave no natural affinity for HALR by modifying their targeting moleculesto include the HALR-binding domain(s) of the Sindbis virus envelopeprotein(s) (preferably, the HALR-binding domain of E2), or animmunoglobilin fragment recognizing HALR, or the IgG-binding domain ofprotein A that can be administered together with anti-HALR antibodies,or laminin (or the HALR binding portion thereof). For example, in aspecific embodiment, the invention provides anti-tumor adenovirus-basedvectors, which have the sequence encoding the terminal knob of thecapsid protein fiber deleted to ablate the natural binding to CAR andother adenovirus receptors, while simultaneously inserting the sequenceresponsible for HALR binding (see, e.g., Alemany et al., Nat.Biotechnol., 2000, 18:723-727). In an alternative embodiment,tumor-specific adenovirus-based vectors are administered together with arecombinantly produced bispecific hybrid adapter protein consisting ofthe amino-terminal extracellular domain of the CAR protein (see, e.g.,Dmitriev et al., J. Virol., 2000, 74:6875-84; Ebbinghaus et al., J.Virol., 2001, 75:480-489) and an HALR-binding domain of Sindbis virusE2, or an immunoglobilin fragment recognizing HALR, or the IgG-bindingdomain of protein A that can be administered together with anti-HALRantibodies. In another embodiment, the invention similarly providesretrovirus-based anti-tumor vectors which have altered targetingproperties due to replacement of the receptor-binding region of envprotein (e.g., N-terminal region of env in murine leukemia retrovirus(MLV)-based vectors) with the HALR-binding domain (see, e.g., Ohno andMeruelo, Biochem. Mol. Med., 1997, 62:123-127).

Taken together, the invention advantageously provides a method fortreating a mammal suffering from a tumor, in which the cells of thetumor express greater levels of HALR compared to normal cells of thesame lineage. The different levels of HALRs result in target-mediateddelivery, i.e., preferential binding of vectors of the invention totumor cells. “Greater levels” of expression generally refer herein tolevels that are expressed by tumor cells (as compared to non-tumorcells) and result in such preferential binding, e.g., at least a 3-foldgreater binding, preferably at least a 30-fold greater binding, mostpreferably at least a 300-fold greater binding. The increased level ofexpression in tumor cells can be evaluated on an absolute scale, i.e.,relative to any other HALR expressing non-tumor cells described, or on arelative scale, i.e., relative to the level expressed by untransformedcells in the same lineage as the transformed cancer cells (e.g.,melanocytes in the case of melanoma; hepatocytes in the case of hepaticcarcinoma; ovarian endothelial cells in the case of ovarianadenocarcinoma, renal endothelial or epithelial cells in the case ofrenal carcinoma).

General Definitions

The terms “vector”, “cloning vector”, “expression vector”, and “helpervector” mean the vehicle by which a DNA or RNA sequence (e.g., a foreigngene) can be introduced into a host cell, so as to promote expression(e.g., transcription and/or translation) of the introduced sequence.Vectors include plasmids, phages, viruses, etc. As used herein withrespect to viral vectors of the invention, “expression vector” is usedmost commonly to refer to a vector that is capable of infecting a hostcell, while the term “helper vector” is used to refer to a vector thatis able to mediate proper packaging of the “expression vector” into avirus-like particle.

As used herein, the term “heterologous sequence or gene” means a nucleicacid (RNA or DNA) sequence, which is not naturally found in associationwith the nucleic acid sequences of the specified molecule, e.g., analphavirus genome. Similarly, the term “heterologous protein or peptide”means a protein, peptide and/or amino acid sequence not naturallyencoded in an alphavirus genome. Within the meaning of the presentinvention, a heterologous gene contained in the aplhavirus-based vectorsis typically of a non-viral origin. However, the term can also includeaphavirus sequences which have been altered by human manipulation tocause changes (e.g., nucleic acid deletions, substitutions, and/oradditions) in the primary nucleic acid sequence and/or positioning inthe native (e.g., naturally occurring) virus molecule. Preferredheterologous genes of the instant invention include, but are not limitedto, reporter genes (such as β-galactosidase and luciferase genes),anti-tumor genes (such as suicide genes, apoptosis-inducing genes, tumorsuppressor genes, oncogene antagonist genes, immunostimulatory genes,tumor suppressor effector genes, antisense oligonucleotide-encodingsequences, ribozyme-encoding sequences, or immunogenic peptide-encodingsequences), and genes encoding tumor-targeting molecules (such as genesencoding chimeric envelope proteins containing a tumor-specificreceptor-binding sequence, sequences encoding peptide mimetics withaffinity for tumor-specific binding sites, genes encoding immunoglobulinmolecules/fragments recognizing tumor-specific antigens, or genesencoding the IgG-binding domain of protein A that can be administeredtogether with anti-viral receptor [e.g., anti-HALR] antibodies).

As used herein, the term “infectious”, when used to describe analphavirus-based RNA molecule, means an RNA molecule which isself-replicating and provides for transcription in a host cell. The term“replication”, when used in conjunction with an alphavirus genomic RNAor a recombinant alphavirus-based vector RNA molecule mean production offull-length equivalents of (+)-strand RNA using (−)-strand RNA as atemplate.

As used herein, the term “transfection” is understood to include anymeans, such as, but not limited to, adsorption, microinjection,electroporation, lipofection and the like for introducing an exogenousnucleic acid molecule into a host cell. The term “transfected” or“transformed”, when used to describe a cell, means a cell containing anexogenously introduced nucleic acid molecule and/or a cell whose geneticcomposition has been altered by the introduction of an exogenous nucleicacid molecule.

As used herein, the term “preferential binding” or “preferentialaffinity” refers to the ability of the viral vector to interact with agiven cellular receptor (e.g., HALR) leading to an increased infectionof cells expressing this receptor. It follows, that vectors of thepresent invention having preferential affinity for HALR receptors wouldparticularly efficiently infect tumor cells which express increasednumbers of HALRs.

As used herein, the term “tumor” refers to a malignant tissue comprisingtransformed cells that grow uncontrollably. Tumors include leukemias,lymphomas, myelomas, plasmacytomas, and the like; and solid tumors.Examples of solid tumors that can be treated according to the inventioninclude sarcomas and carcinomas such as, but not limited to:fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, epidermoid carcinoma, adenocarcinoma, sweat glandcarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervicalcancer, testicular tumor, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, neuroglioma, and retinoblastoma. As notedabove, the method of the invention depends on expression of HALRs bycells of the tumor targeted for treatment.

The term “tumor-specific cellular determinant” or “tumor-specifictarget” is used herein to broadly define any molecule on the surface ofa tumor cell, which can be used for selective or preferential targetingof this cell by the vectors of the invention. Tumor-specific cellulardeterminants for the vectors of the instant invention include withoutlimitation any tumor cell surface protein, peptide, oligonucleotide,lipid, polysaccharide, and a small molecule ligand. Preferredtumor-specific cellular determinants of the invention are tumor-specificmembrane proteins such as ErbB receptors, Melan A [MART1], gp100,tyrosinase, TRP-1/gp 75, and TRP-2 (in melanoma); MAGE-1 and MAGE-3 (inbladder, head and neck, and non-small cell carcinoma); HPV EG and E7proteins (in cervical cancer); Mucin [MUC-1] (in breast, pancreas,colon, and prostate cancers); prostate-specific antigen [PSA] (inprostate cancer); carcinoembryonic antigen [CEA] (in colon, breast, andgastrointestinal cancers), LH/CG receptor (in choriocarcinoma), and suchshared tumor-specific antigens as MAGE-2, MAGE-4, MAGE-6, MAGE-10,MAGE-12, BAGE-1, CAGE-1,2,8, CAGE-3 to 7, LAGE-1, NY-ESO-1/LAGE-2,NA-88, GnTV, and TRP2-INT2, etc. HALRs as well as other determinants(e.g., EGF receptors or α_(v)β₃ integrins), which are expressed athigher levels on the surface of certain tumor cells, as compared tonormal cells of the same lineage, are also encompassed by the term“tumor-specific cellular determinant.”

As used herein, the term “mammal” has its ordinary meaning, andspecifically includes primates, and more specifically includes humans.Other mammals that may be treated for the presence of a tumor include,but are not limited to, canine, feline, rodent (racine, murine, lupine,etc.), equine, bovine, ovine, caprine, and porcine species.

The term “subject” as used herein refers to a vertebrate, preferably amammal (e.g., rodent such as mouse). In particular, the term refers tohumans.

The term “about” or “approximately” usually means within an acceptableerror range for the type of value and method of measurement. Forexample, it can mean within 20%, more preferably within 10%, and mostpreferably still within 5% of a given value or range. Alternatively,especially in biological systems, the term “about” means within about alog(i.e., an order of magnitude) preferably within a factor of two of agiven value.

The term “treat” is used herein to mean to relieve or alleviate at leastone symptom of a disease in a subject. Within the meaning of the presentinvention, the term “treat” may also mean to prolong the prepatency,i.e., the period between infection and clinical manifestation of adisease. The term “protect” is used herein to mean prevent or treat, orboth, as appropriate, development or continuance of a disease in asubject. Within the meaning of the present invention, the disease iscancer.

The phrase “pharmaceutically acceptable”, as used in connection withcompositions of the invention, refers to molecular entities and otheringredients of such compositions that are physiologically tolerable anddo not typically produce untoward reactions when administered to ahuman. Preferably, as used herein, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in mammals, and more particularly inhumans.

The term “therapeutically effective” applied to dose or amount refers tothat quantity of a compound or pharmaceutical composition that issufficient to result in a desired activity upon administration to amammal in need thereof. As used herein with respect to viral vectors ofthe invention, the term “therapeutically effective amount/dose” refersto the amount/dose of a vector or pharmaceutical composition containingthe vector that is sufficient to produce an effective anti-tumorresponse upon administration to a mammal.

The term “antibody” is used in the broadest sense and specificallycovers not only native antibodies but also single monoclonal antibodies(including agonist and antagonist antibodies), antibody compositionswith polyepitopic specificity, as well as antibody fragments (e.g., Fab,F(ab′)₂, scFv and Fv), so long as they exhibit the desired biologicalactivity.

The term “innate immunity” or “natural immunity” refers to innate immuneresponses that are not affected by prior contact with the antigen. Theprotective mechanisms of the innate immunity include, among others,natural killer (NK) cells, which destroy microbes and certain tumorcells, and attack certain virus infected cells, and the inflammatoryresponse, which mobilizes leukocytes such as macrophages and dendriticcells to phagocytose invaders.

Vectors

Most preferred vectors of the invention are alphavirus-based vectors, inparticular, replication defective naturally targeted Sindbis virus-basedvectors. Other preferred vectors of the invention in vitro, in vivo, andex vivo are viral vectors, such as retroviruses (includinglentiviruses), herpes viruses, adenoviruses, adeno-associated viruses,vaccinia virus, papillomavirus, Epstein Barr virus (EBV), baculovirus,and other recombinant viruses with or modified to have the desirablecellular tropism, i.e., binding preferentially to the high affinitylaminin receptor (HALR).

Methods for constructing and using viral vectors are known in the art(see, e.g., Miller and Rosman, BioTechniques 1992, 7:980-990). Inaccordance with the present invention there may be employed conventionalmolecular biology, microbiology, and recombinant DNA techniques withinthe skill of the art. Such techniques are well-known and are explainedfully in the literature. See, e.g., Sambrook, Fritsch and Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

Various companies produce viral vectors commercially, including but byno means limited to Avigen, Inc. (Alameda, Calif.; AAV vectors), CellGenesys (Foster City, Calif.; retroviral, adenoviral, AAV vectors, andlentiviral vectors), Clontech (retroviral and baculoviral vectors),Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors), Genvec(adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviralvectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpesviral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford,United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France;adenoviral, vaccinia, retroviral, and lentiviral vectors).

Preferably, the viral vectors of the invention are replicationdefective, that is, they are unable to replicate autonomously in thetarget cell. Preferably, the replication defective virus is a minimalvirus, i.e., it retains only the sequences of its genome which arenecessary for target cell recognition and encapsidating the viralgenome. Replication defective virus is not infective after introductioninto a cell. Use of replication defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Thus, a specific tissue can bespecifically targeted. In addition to replication defective alphavirusvectors, examples of particular vectors include, but are not limited to,defective herpes virus vectors (see, e.g., Kaplitt et al., Molec. Cell.Neurosci. 1991, 2:320-330; Patent Publication RD 371005 A; PCTPublications No. WO 94/21807 and WO 92/05263), defective adenovirusvectors (see, e.g., Stratford-Perricaudet et al., J. Clin. Invest. 1992,90:626-630; La Salle et al., Science 1993, 259:988-990; PCT PublicationsNo. WO 94/26914, WO 95/02697, WO 94/28938, WO 94/28152, WO 94/12649, WO95/02697, and WO 96/22378), and defective adeno-associated virus vectors(Samulski et al., J. Virol. 1987, 61:3096-3101; Samulski et al., J.Virol. 1989, 63:3822-3828; Lebkowski et al., Mol. Cell. Biol. 1988,8:3988-3996; PCT Publications No. WO 91/18088 and WO 93/09239; U.S. Pat.Nos. 4,797,368 and 5,139,941; European Publication No. EP 488 528).

As specified above, various strategies can be implemented to targetvectors to the HALR, including but not limited to pseudotyping the viralvector by introducing an HALR binding sequence, e.g., a Sindbis virus E2protein, into the virus; by modifying viral capsid or envelope proteinsto contain an HALR binding sequence; by using a bi-specific reagent thatbinds to the vector and to HALR; or using combinations of theseapproaches.

Adenovirus-based vectors. Adenoviruses are eukaryotic DNA viruses thatcan be modified to efficiently deliver a nucleic acid of the inventionto a variety of cell types. Various serotypes of adenovirus exist. Ofthese serotypes, preference is given, within the scope of the presentinvention, to using type 2 or type 5 human adenoviruses (Ad 2 or Ad 5)or adenoviruses of animal origin (see PCT Publication No. WO94/26914).Those adenoviruses of animal origin which can be used within the scopeof the present invention include adenoviruses of canine, bovine, murine(e.g., Mav1 [Beard et al., Virology, 1990, 75:81]), ovine, porcine,avian, and simian (e.g., SAV) origin. Preferably, the adenovirus ofanimal origin is a canine adenovirus, more preferably a CAV2 adenovirus(e.g., Manhattan or A26/61 strain [ATCC Accession No. VR-800]). Variousreplication defective adenovirus and minimum adenovirus vectors havebeen described (PCT Publications No. WO94/26914, WO95/02697, WO94/28938,WO94/28152, WO94/12649, WO95/02697, WO96/22378). The replicationdefective recombinant adenoviruses according to the invention can beprepared by any technique known to the person skilled in the art(Levrero et al., Gene, 1991, 101:195; EP Publication No. 185 573;Graham, EMBO J., 1984, 3:2917; Graham et al., J. Gen. Virol., 1977,36:59). Recombinant adenoviruses are recovered and purified usingstandard molecular biological techniques, which are well known to one ofordinary skill in the art.

Adeno-associated virus-based vectors. The adeno-associated viruses (AAV)are DNA viruses of relatively small size which can integrate, in astable and site-specific manner, into the genome of the cells which theyinfect. They are able to infect a wide spectrum of cells withoutinducing any effects on cellular growth, morphology or differentiation,and they do not appear to be involved in human pathologies. The AAVgenome has been cloned, sequenced and characterized. The use of vectorsderived from the AAVs for transferring genes in vitro and in vivo hasbeen described (see PCT Publications No. WO 91/18088 and WO 93/09239;U.S. Pat. Nos. 4,797,368 and 5,139,941; EP Publication No. 488 528). Thereplication defective recombinant AAVs according to the invention can beprepared by cotransfecting a plasmid containing the nucleic acidsequence of interest flanked by two AAV inverted terminal repeat (ITR)regions, and a plasmid carrying the AAV encapsidation genes (rep and capgenes), into a cell line which is infected with a human helper virus(e.g., an adenovirus). The AAV recombinants which are produced are thenpurified by standard techniques.

Retroviral vectors. In another embodiment, the invention providesretroviral vectors, e.g., as described in Mann et al., Cell 1983,33:153; U.S. Pat. Nos. 4,650,764, 4,980,289, 5,124,263, and 5,399,346;Markowitz et al., J. Virol. 1988, 62:1120; EP Publications No. 453 242and 178 220; Bernstein et al. Genet. Eng. 1985, 7:235; McCormick,BioTechnology 1985, 3:689; and Kuo et al., 1993, Blood, 82:845. Theretroviruses are integrating viruses which infect dividing cells. Theretrovirus genome includes two LTRs, an encapsidation sequence and threecoding regions (gag, pol and env). Replication defective non-infectiousretroviral vectors are manipulated to destroy the viral packagingsignal, but retain the structural genes required to package theco-introduced virus engineered to contain the heterologous gene and thepackaging signals. Thus, in recombinant replication defective retroviralvectors, the gag, pol and env genes are generally deleted, in whole orin part, and replaced with a heterologous nucleic acid sequence ofinterest. These vectors can be constructed from different types ofretroviruses, such as HIV (human immuno-deficiency virus), MoMuLV(murine Moloney leukaemia virus), MSV (murine Moloney sarcoma virus),HaSV (Harvey sarcoma virus), SNV (spleen necrosis virus), RSV (Roussarcoma virus), and Friend virus. Suitable packaging cell lines havebeen described in the prior art, in particular, the cell line PA317(U.S. Pat. No. 4,861,719); the PsiCRIP cell line (PCT Publication No. WO90/02806) and the GP+envAm-12 cell line (PCT Publication No. WO89/07150). In addition, recombinant retroviral vectors can containmodifications within the LTRs for suppressing transcriptional activityas well as extensive encapsidation sequences which may include a part ofthe gag gene (Bender et al., J. Virol. 1987, 61:1639). Recombinantretroviral vectors are purified by standard techniques known to thosehaving ordinary skill in the art.

Retrovirus vectors can also be introduced by DNA viruses, which permitsone cycle of retroviral replication and amplifies tranfection efficiency(see PCT Publications No. WO 95/22617, WO 95/26411, WO 96/39036, WO97/19182).

In a specific embodiment of the invention, lentiviral vectors can beused as agents for the direct delivery and sustained expression of atransgene in several tissue types, including brain, retina, muscle,liver, and blood. This subtype of retroviral vectors can efficientlytransduce dividing and nondividing cells in these tissues, and maintainlong-term expression of the gene of interest (for a review, see,Naldini, Curr. Opin. Biotechnol. 1998, 9:457-63; Zufferey, et al., J.Virol. 1998, 72:9873-80). Lentiviral packaging cell lines are availableand known generally in the art (see, e.g., Kafri, et al., J. Virol.,1999, 73: 576-584).

Non-viral vectors. In another embodiment, the invention providesnon-viral vectors that can be introduced in vivo, provided that thesevectors contain a targeting peptide, protein, antibody, etc. thatspecifically binds HALR. For example, compositions of synthetic cationiclipids, which can be used to prepare liposomes for in vivo transfectionof a vector carrying an anti-tumor therapeutic gene, are described inFelgner et. al., Proc. Natl. Acad. Sci. USA 1987, 84:7413-7417; Felgnerand Ringold, Science 1989, 337:387-388; Mackey, et al., Proc. Natl.Acad. Sci. USA 1988, 85:8027-8031; and Ulmer et al., Science 1993,259:1745-1748. Useful lipid compounds and compositions for transfer ofnucleic acids are described, e.g., in PCT Publications No. WO 95/18863and WO96/17823, and in U.S. Pat. No. 5,459,127. Targeting peptides,e.g., laminin or HALR-binding laminin peptides, and proteins such asanti-HALR antibodies, or non-peptide molecules can be coupled toliposomes covalently (e.g., by conjugation of the peptide to aphospholipid or cholesterol; see also Mackey et al., supra) ornon-covalently (e.g., by insertion via a membrane binding domain ormoiety into the bilayer membrane).

Alphavirus, Particularly, Sindbis Virus Vectors

Alphaviruses are well known in the art, and include without limitationEquine Encephalitis viruses, Semliki Forest virus and related species,Sindbis virus, and recombinant or ungrouped species (see Strauss andStrauss, Microbiol. Rev. 1994, 58:491-562, Table 1, p. 493).

Preferably, the alphaviral vectors of the invention are prepared as areplication defective virus. As used herein the term “replicationdefective virus” has its ordinary meaning, i.e., a virus that ispropagation incompetent as a result of modifications to its genome.Thus, once such recombinant virus infects a cell, the only course it canfollow is to express any viral and heterologous protein contained in itsgenome, and, in the case of Sindbis and other alphavirus vectors, induceapoptosis. In a specific embodiment, the replication defectivealphavirus-based vectors of the invention contain genes encodingnonstructural proteins, and are self-sufficient for RNA transcriptionand gene expression. However, these vectors lack genes encodingstructural proteins, so that a helper genome is needed to allow them tobe packaged into infectious particles. In addition to providingtherapeutically safe vectors, the removal of the structural proteinsincreases the capacity of these vectors to incorporate more than 6 kb ofheterologous sequences. In another embodiment, propagation incompetenceof the alphavirus vectors of the invention is achieved indirectly, e.g.,by removing the packaging signal which allows the structural proteins tobe packaged in virions being released from the packaging cell line.

Because Sindbis virus does not pose a significant health hazard, it canbe also used in a propagation-competent form. In other words, unlikemost viral vectors, it is not essential to make Sindbis virus vectorsdefective or replication incompetent, although this is preferred forrigorous safety reasons. Accordingly, the invention contemplates bothpropagation defective and propagation competent Sindbis virus vectors.

As noted above, alphaviruses, particularly Sindbis virus vectors,naturally induce apoptosis, also called “programmed cell death”.Apoptosis is an intrinsic cellular process that leads to nucleardestruction, digestion of DNA, and ultimately cell necrosis andablation. The apoptotic potential of a vector of the invention can betested in vitro using cells in tissue culture (e.g., any of the hostcells discussed in Example 1, infra).

Various methods for the preparation of alphavirus vectors of theinvention are known in the art. In the case of Sindbis virus-basedvectors, in general, vector preparation involves co-transfection of apackaging cell line with a capped mRNA containing Sindbis non-structuralgenes and, optionally, a heterologous gene under control of a Sindbissubgenomic promoter) and a helper plasmid vector that expresses Sindbisstructural genes (see FIG. 1). The capped mRNA can be produced by invitro transcription.

Various cell lines can be used as packaging cells. These includemammalian cell lines such as CHO (Chinese hamster ovary), BHK (babyhamster kidney), HuH7 (human hepatocellular carcinoma), and the like.One drawback of these cell lines is that production of Sindbis virusvectors induces apoptosis, resulting in destruction of the packagingcell within hours or a few days of co-transfection. Thus, new packagingcells must be prepared on an ongoing basis. In addition, Sindbisvirus-based vectors produced in mammalian cells appear to efficientlyinteract with the negatively charged glucosaminoglycan heparan sulfateleading to increased viral clearence and decreased infectivity (see,e.g., Byrnes and Griffin, 2000, J. Virol., 74:644-651).

To avoid these problems, the present inventors have developed a Sindbisvirus packaging cell line derived from insect cells, preferably theC6/36 mosquito cells (see co-owned, co-pending PCT Application No.PCT/US02/09431, [Attorney Internal Reference No. 5986/2H995-WO0], filedon even date herewith, entitled “Packaging Cell Lines for the ContinuousProduction of Alphavirus Vectors”, based on U.S. Provisional PatentApplication Ser. No. 60/279,048, filed Mar. 27, 2001, both of which arespecifically incorporated herein by reference in its entirety). Togenerate the insect packaging cell line, the present inventors havestably transformed C6/36 cells with two DNA vectors, one encoding thenonstructural genes nsp 1-4 for replicating viral RNA and another(helper) encoding the structural proteins (capsid protein C, E1, E2, E3,and 6K) and the packaging signal. As unexpectedly discovered by thepresent inventors, these insect-derived packaging cells aresubstantially resistant to the apoptosis-induced properties of Sindbisvectors and can be engineered to establish long-term vector producingcultures which give rise to consistently high virus titers. In additionto C6/36 mosquito cells, non-limiting examples of other insect celllines for use in the present invention include u4.4 cells, High Five™cells, Schneider's Drosophila cell line 2, Spodoptera frugiperda SF9cells, and C7-10 cells.

In each case, alphavirus, particularly Sindbis virus, particlescontaining the replication defective viral genome can be harvested fromthe packaging cell culture supernatants, concentrated and purified byany of the techniques known in the art. These techniques include, butare not limited to, centrifugation and ultracentrifugation;chromatography on ion exchange, size exclusion, hydrophobic interaction,hydrophilic interaction, or other types of columns; filtration andultrafiltration; affinity purification; or various other techniquesknown in the art. Isolated viral particles of the invention can beeither preserved in a solution or, preferably, lyophilized and preservedin a form of a powder.

Anti-tumor Therapeutic Genes

The therapeutic vectors of the invention can carry an anti-tumortherapeutic gene, as disclosed herein. In particular, becausealphavirus, particularly Sindbis virus, vectors of the invention cancarry a therapeutic gene payload, they may be modified to include any ofthe gene therapies described below.

The term “anti-tumor gene therapy” as used herein refers to a genetherapy targeted to a tumor, which causes tumor necrosis, apoptosis,growth regulation, i.e., regression or suppression of the tumor.Examples of anti-tumor gene therapies include, but are by no meanslimited to, introduction of a suicide gene, introduction of an apoptosisgene, introduction of a tumor suppressor gene, and introduction of anoncogene antagonist gene. Preferably anti-tumor genes are supplementedwith immunostimulatory genes to enhance recruitment and activation ofimmune effector cells, including mobilized dendritic cells, to thetumor.

Thus, “gene therapy” specifically refers to transfer of a gene encodingan effector molecule into cells, in this case, into tumor cells.

Suicide gene therapies. Introduction of genes that encode enzymescapable of conferring to tumor cells sensitivity to chemotherapeuticagents (suicide gene) has proven to be an effective anti-tumor genetherapy. The present invention provides a method of treating cancer inpart by introducing a gene vector, encoding a protein capable ofenzymatically converting a prodrug, i.e., a non-toxic compound, into atoxic compound. In the method of the present invention, the therapeuticnucleic acid sequence is a nucleic acid coding for a product, whereinthe product causes cell death by itself or in the presence of otherdrugs. A representative example of such a therapeutic nucleic acid isone which codes for thymidine kinase of herpes simplex virus (HSV-tk).Additional examples are thymidine kinase of varicella zoster virus(VZV-tk) and the bacterial gene cytosine deaminase.

The prodrug useful in the methods of the present invention is any thatcan be converted to a toxic product, i.e., toxic to tumor cells.Representative examples of such a prodrug is ganciclovir, which isconverted in vivo to a toxic compound by HSV-tk (Chen et. al., CancerRes. 1996, 56: 3758-3762). Other representative examples of prodrugsinclude acyclovir, FIAU[1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodouracil],6-methoxypurine arabinoside (converted by VZV-tk), and 5-fluorocytosine(converted by cytosine deaminase).

Prodrugs, may be readily administered to a patient by a physician havingordinary skill in the art. Using methods known in the field, suchphysician would also be able to determine the most appropriate dose androute for the administration of the prodrug. For example, ganciclovir ispreferably administered in a dose of from about 1-20 mg/day/kg bodyweight; acyclovir is administered in a dose of from about 1-100mg/day/kg body weight, and FIAU is administered in a dose of from about1-50 mg/day/kg body weight.

Apoptosis-inducing, Anti-oncogene, and Tumor Suppressor Gene Therapies.

Tumor initiation and progression in many cancer types are linked tomutations in oncogenes (e.g., ras, myc) and tumor suppressor genes(e.g., Rb, p53). A number of approaches are being pursued usinganti-oncogene and/or tumor suppressor effector molecules includingmonoclonal and single chain antibodies, antisense oligonucleotides,ribozymes, analogs, and immunogenic peptides (Chen, Mol. Med. Today1997, 3:160-167; Spitz, et al., Anticancer Res. 1996, 16:3415-3422;Indolfi et al., Nat. Med. 1996, 2:634-635; Kijima et al., Pharmacol.Ther. 1995, 68:247-267; PCT Publications No. WO 94/24297 and WO97/16547; French Patent Application No. FR 08729). These moleculesspecifically suppress tumor growth and increase the apoptosis rate intumor cells. Their mechanisms of action require constant presence ofsuppressor or anti-oncogene molecules for sustained responses, however,they have not been shown to induce tumor-specific immunity, which hasthe potential of memory necessary for protection against the recurrenceof the disease. Combination of these tumor growth-specific strategieswith immunostimulatory therapies will have a synergistic effects ontumor regression and induction of protective immune response.

Immunostimulatory therapies. The invention also provides for immune cellstimulation, such as dendritic cell (DC) mobilization, to generate astrong anti-tumor immune response. The term “dendritic cell (DC)mobilizing agent” refers to a molecule that activates DC functionalactivity. A well-known DC mobilizing agent is flt-3 ligand (flt-3L).Anti-tumor-based immunotherapy efficacy can be enhanced by the additionof a variety of cytokines. Cytokines such as IL-12 amplify the antigenpresenting and immunomodulatory capabilities of DC and inhibit tumorangiogenesis, which consecutively can induce immune susceptibility ofthe tumor. Conversely, cytokines such as IL-7 may induce more potent Tcell responses and effectively reverse T cell defects in vivo. Asdisclosed herein, other cytokines, such as granuclocyte-macrophagecolony stimulating factor (GM-CSF), IL-2, IL-3, IL-4, TNF-α, and c-kitligand can be also used in combination with the DC mobilizing agent.

These cytokines can be administered, e.g., as soluble or microparticleencapsulated protein or by introducing the gene in viral or non-viralvectors including the vectors of the present invention. Systemicdelivery of such cytokines along with local anti-tumor gene therapiesmay increase the tumor distribution of these cytokines, which may berequired for long term reversal of T cell defects and effective tumorresponses. These cytokines, depending on the mode of administration, mayhave a critical role in exploiting the immune inflammation for anefficient anti-tumor immune response.

Tumor growth inhibitors. The term “tumor growth inhibitor” is usedherein to refer to a protein that inhibits tumor growth, such as but notlimited to interferon (IFN)-γ, tumor necrosis factor (TNF)-α, TNF-β, andsimilar cytokines. Alternatively, a tumor growth inhibitor can be anantagonist of a tumor growth factor. Such antagonists include, but arenot limited to, antagonists of tumor growth factor (TGF)-β and IL-10.The present invention contemplates administration of tumor growthinhibitor proteins systemically, or alternatively by gene therapy.

Anti-angiogenic factors. Tumor angiogenesis is an integral part of tumorprogression and a variety of therapies targeted to inhibit angiogenesisare under development as cancer therapies. Anti-angiogenesis therapiesprimarily reverse the growth/apoptosis balance of the tumor and inducedormancy. Once the administration of these therapies is halted,angiogenesis can resume and tumor growth progresses. Anti-angiogenesisis a powerful mechanism to specifically reduce the bulk of the tumorwithout adverse side effects in patients. The dormancy therapy inducedby anti-angiogenesis paves the way for other therapy schemes to succeedby debulking the tumor, altering the tumor microenvironment, eliminatingthe immunosuppressive effects, and making the tumor more susceptible forimmune-mediated clearance.

An “anti-angiogenic factor” is a molecule that inhibits angiogenesis,particularly by blocking endothelial cell migration. Such factorsinclude without limitation fragments of angiogenic proteins that areinhibitory (such as the amino terminal fragment of urokinase [PCTPublication No. WO 93/15199]); angiostatin (O'Reilly et al., Cell 1994,79:315-328); endostatin; soluble forms of receptors for angiogenicfactors, such as urokinase receptor or FGF/VEGF receptor (Wilhem et al.,FEBS Letters 1994, 337:131-134); molecules which block endothelial cellgrowth factor receptors (O'Reilly et. al. Cell 1997, 88:277-285;O'Reilly, Nat. Med. 1996, 2:689-692), and Tie-1 or Tie-2 inhibitors. Thepresent invention contemplates administration of anti-angiogenesisfactors systemically, or alternatively by gene therapy. Preferably, ananti-angiogenic factor for use in the invention is a protein orpolypeptide, which is encoded by a gene contained in the vectors of theinvention.

Vector Therapy

As noted above, the alphavirus-based vectors, and particularly Sindbisvirus-based vectors, of the invention can be used to treat variouscancers. Also, the non-alphavirus-based vectors of the invention can beused to treat any cancer in which tumor cells express increased levelsof HALR.

Isolated, preferably purified viral vector obtained as described abovecan be formulated in a pharmaceutical composition for administration toa patient. As used herein, a “pharmaceutical composition” includes theactive agent, i.e., the viral vector, and a pharmaceutically acceptablecarrier, excipient, or diluent. The phrase “pharmaceutically acceptable”refers to molecular entities and compositions that are physiologicallytolerable and do not typically produce an allergic or similar untowardreaction, such as gastric upset, dizziness and the like, whenadministered to a human. Preferably, as used herein, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the compound is administered.Such pharmaceutical carriers can be sterile liquids, such as water andoils, including those of petroleum, animal, vegetable or syntheticorigin, such as peanut oil, soybean oil, mineral oil, sesame oil and thelike. Water or aqueous solution saline solutions and aqueous dextroseand glycerol solutions are preferably employed as carriers, particularlyfor injectable solutions. Suitable pharmaceutical carriers are describedin “Remington's Pharmaceutical Sciences” by E. W. Martin.

For human therapy, the viral vectors will be prepared in accordance withgood manufacturing process (GMP) standards, as set by the Food & DrugAdministration (FDA). Quality assurance (QA) and quality control (QC)standards will include testing for replication competent virus (if thevirus vector is replication defective), activity (colony forming units[CFU] per number of viral particles, tested by induction of apoptosis orcytopathic effect (CPE), or by expression of a marker gene such asβ-galactosidase), toxicity, and other standard measures.

In order to treat the tumor cells, the pharmaceutical composition isadministered by any route that will permit homing of the vector to thetumor cells. Preferably, administration is parenteral, including, butnot limited to, intravenous, intra-arteriole, intramuscular,intradermal, subcutaneous, intraperitoneal, intraventricular, andintracranial administration. As disclosed herein, viral vectors can bealso administered to the tumor-bearing animal via intranasal or oralroute (see Hardy, In: The Arbiviruses: Epidemiology and Ecology, Ch. 4,pp. 87-126). Importantly, however, in contrast to other viral vectors ingene therapy, administration of alphavirus and non-alphavirusHALR-targeted vectors of the invention need not be locally to the tumor.Indeed, one of the advantages of this invention is the high specificityand affinity of the vector for cancer cells, even micrometastases thatcannot be resected or located by standard techniques (e.g., CATscanning, MRI scanning, etc.).

In therapeutic treatments of the invention, a therapeutically effectiveamount of the vector is administered to a patient. As used herein, theterm “therapeutically effective amount” means an amount sufficient toreduce by at least about 15 percent, preferably by at least 50 percent,more preferably by at least 90 percent, and most preferably prevent, aclinically significant deficit in the activity, function and response ofthe host. Alternatively, a therapeutically effective amount issufficient to cause an improvement in a clinically significant conditionin the host. Specifically, a therapeutically effective amount will causeone or more of the following: apoptosis of tumor cells; necrosis oftumor cells; elimination or prevention of tumor metastases; reduction inthe rate of tumor growth; reduction in tumor size or tumor shrinkage;scab formation over cutaneous tumor; elimination of the tumor; remissionof the cancer; an increase in the time for reappearance of the cancer;and increased time of survival of the patient. The frequency and dosageof the vector therapy can be titrated by the ordinary physician usingstandard dose-to-response techniques, but generally will be in the rangeof from 10⁶ to 10¹² viral particles per dose administered daily, weekly,biweekly, or monthly at least twice and preferably at least three times.

Combination Therapies

Vaccines. In order to increase the tumor antigen specific immuneresponse, one could introduce defined tumor-associated antigens (TAA) inthe system to specifically increase the level of antigen. These TAAcould be introduced, e.g., as proteins, peptides or as heterologousgenes encoded by the viral vectors of the invention. Immunization withthese antigens could either follow or occur during DC mobilization andanti-tumor gene therapy schemes. Essentially, this strategy enhances aneffective immune response against specific antigen in conjunction withoverall immune response. Specific immunization may lead to theexpression of an immune enhancing cytokine milieu which can promote theresponse against the antigens released by the tumor necrosis. Suchimmunization could be combined with immune activating cytokines (proteinor genes) to further enhance the effects.

Besides the defined antigen-based vaccines, a number of vaccinestrategies are being explored in the laboratory as well as in theclinic. One well researched strategy in animal models is themodification of autologous or allogeneic tumor cell using cytokine genes(e.g., IL-2, GM-CSF, IL-12, IL-4) as well as some key costimulatorymolecule genes (e.g., B7.1, B7.2). These gene modified tumor vaccinesprove the concept of breaking peripheral tolerance and anergy usingimmunological mechanisms (Clary et al. Cancer Gene Ther. 1997, 4:97-104;Gilboa, Semin. Oncol. 1996, 23:101-107). Other similar approachesinclude use of tumor lysates, proteins, or RNA pulsed DC and fusion oftumor cells with DC to induce a potent tumor immune response. All theseapproaches have a common theme, which is the delivery of antigenicmolecules to the DC to induce efficient processing and presentation ofthese antigens to T cells. Since these schemes are based on theavailability of DC, DC mobilization is expected to amplify the effectsobserved with these approaches.

Chemotherapeutic agents, radiation, and surgery (tumor resection).Although the methods of the invention are effective in inhibiting tumorgrowth and metastasis, the vectors and methods of the present inventionare advantageously used in conjunction with other treatment modalities,including without limitation surgery, radiation, chemotherapy, and othergene therapies. For example, the vectors of the invention can beadministered in combination with nitric oxide inhibitors, which havevasoconstrictive activity and reduce blood flow to the tumor. In anotherembodiment, a vector of the invention can be administered with achemotherapeutic such as, though not limited to, taxol, taxotere andother taxoids (e.g., as disclosed in U.S. Pat. Nos. 4,857,653;4,814,470; 4,924,011, 5,290,957; 5,292,921; 5,438,072; 5,587,493;European Patent No. 0 253 738; and PCT Publications No. WO 91/17976, WO93/00928, WO 93/00929, and WO 96/01815), or other chemotherapeutics,such as cis-platin (and other platin intercalating compounds), etoposideand etoposide phosphate, bleomycin, mitomycin C, CCNU, doxorubicin,daunorubicin, idarubicin, ifosfamide, and the like.

EXAMPLES

The following examples illustrate the invention without limiting itsscope.

Example 1 Sindbis Vectors Mediate Potent Anti-Tumor Activity Materialsand Methods

Cell lines. Babyhamsterkidney (BHK-21, ATCC Accession No. CCL-10) andovarian carcinoma (ES-2, ATCC Accession No. CRL-1978) cells wereobtained from the American Type Culture Collection (ATCC, Manassas, Va.)and maintained in minimum essential α-modified media (αMEM, JRHBioscience, Lenexa, Kans.) supplemented with 5% fetal bovine serum (FBS,Gemini Bioproducts, Inc., Calabasas, Calif.). A human hepatocellularcarcinoma cell line HuH7 was obtained from Dr. H. Yamamoto (HyogoCollege of Medicine, Japan) and maintained in Dulbecco's modifiedEagle's medium (DMEM, JRH Bioscience, Lenexa, Kans.) supplemented with10% FBS. HT29 human colorectal adenocarcinoma cells (ATCC Accession No.HTB-38) were obtained from the ATCC and maintained in McCoy's 5A medium(Iwakata & Grace modification, Mediatech, Inc, Herndon, Va.) with 10%FBS. CFPAC-1 pancreatic cancer cells (ATCC Accession No. CRL-1918),SKOV-3 ovarian adenocarcinoma cells (ATCC Accession No. HTB-77), andA431 epidermoid carcinoma cells (ATCC Accession No. CRL-2592) wereobtained from the ATCC and maintained in DMEM/low modified (JRHBioscience, Lenexa, Kans.) with 10% FBS. A498 renal cancer cells (ATCCAccession No. HTB-44), HT1197 bladder cancer cells (ATCC Accession No.CRL-1473), and LS174T colon carcinoma cells (ATCC Accession No. CL-188)were obtained from the ATCC and were maintained in minimum essentialmedium Eagle with Earle's salts and L-glutamine (Mediatech Inc.,Herndon, Va.) supplemented with 10% FBS. All basal media above weresupplemented with 100 mg/mL of penicillin-streptomycin (Mediatech, Inc.,Herndon, Va.) and 0.5 mg/ml of Amphotericin B (Mediatech, Inc., Herndon,Va.).

Vectors. FIG. 1 shows schematic representation of the Sindbisvirus-derived expression and helper vectors (see also Bredenbeek et al.,J. Virol., 1993, 67:6439-6446, Invitrogen Co., Carlsbad, Calif.). ASindbis virus-based expression vector SinRep/LacZ encodes the viralpackaging signal, nonstructural proteins nsp1-4 essential forreplicating the RNA transcript, the viral promoter for subgenomictranscription, and the LacZ reporter gene. A helper plasmid DH-BBencodes the Sindbis structural genes, i.e., capsid (C), E3, E2, 6K, andE1, which are necessary for viral packaging. Not shown in FIG. 1 are thetwo other Sindbis virus-based expression vectors used in the presentExample, SinRep/Luc and SinRep/IL12. To construct the SinRep/Luc vector,a DNA fragment containing the firefly luciferase gene (Luc) was excisedfrom the Nhe I site and Xba I site of the pGL3 plasmid (Promega Co.,Madison, Wis.) and subcloned into the Xba I site of SinRep vector(Invitrogen Co., Carlsbad, Calif.). To construct the SinRep/IL12 vector,a Sindbis vector containing two subgenomic promoters (SinRep/2P_(SG))was first constructed by the insertion of a second subgenomic promoterDNA into the multiple cloning site (MCS) downstream of the originalsubgenomic promoter. The deoxyoligonucleotide containing the P_(SG)sequence 5′-CGCGTAAA GCATCTCTACGGTGGTCCTAATAGTGCATG-3′ (SEQ ID NO: 1)was annealed to its complementary sequence5′-CACTATTAGGACCACCGTCGAGATGCTTTA-3′ (SEQ ID NO: 2) before ligation tothe SinRep plasmid digested with Mlu I and Sph I. The murine IL12α-subunit gene (mP35, ATCC Accession No. 87596) and the IL12 β-subunitgene (mP40, ATCC Accession No. 87595) were subcloned into the Mlu I siteand the Stu I site (downstream of the Sph I site) of SinRep/2P_(SG),respectively, and the final construct was named SinRep/IL12.

In vitro transcription and transfection for Sindbis virus production.Plasmids for in vitro transcription were prepared with the QIAGENplasmid kit (QIAGEN, Chatsworth, Calif.). Plasmids DH-BB, SinRep/LacZ,and SinRep/IL12 were linearized using XhoI restriction enzyme. PlasmidSinRep/Luc was linerized using Not I restriction enzyme, since the Lucgene contains an internal Xho I site. Linearized plasmids were furtherpurified by phenol/chloroform extraction followed by ethanolprecipitation. In vitro transcription reactions, from the SP6 promoter,were carried out by using the InvitroScript Cap Kit (Invitrogen Co.,Carlsbad, Calif.) or mMESSAGE mMACHINE™ high yield capped RNAtranscription kit (Ambion Inc., Austin, Tex.) to produce largequantities of capped mRNA transcripts. The quality of mRNA was checkedon 1% agarose gels. For co-transfection of helper DH-BB and eitherSinRep/LacZ, SinRep/Luc, or SinRep/IL12 into BHK cells, electroporationswere performed as described before (Ohno et al., Nature Biotechnol.,1997, 15: 763-767). Electroporated cells were transferred into 10 ml ofαMEM containing 5% FBS and incubated for 12 hr. Cells were then washedwith phosphate-buffered saline (PBS) and incubated in 10 ml of Opti-MEMI medium (GIBCO-BRL) without FBS. After 24 hr, culture supernatants wereharvested and aliquots were stored at −80° C.

Infection Assay and virus quantification. Viral supernatant dilutions(300 ml) were added to 2×10⁵ cells in 12-well plates. After 1 hrincubation at room temperature, cells were washed with PBS and incubatedwith medium for 24 hrs. Viral infection was evaluated by X-gal staining:infected cells were fixed in PBS containing 0.5% glutaraldehyde for 20min followed by washing with PBS three times; cells were then stainedwith PBS containing 1 mg/ml X-gal(5-Bromo-4-chloro-β-D-galactropyranoside, Fisher Scientific), 5 mMpotassium ferricyanide, 5 mM potassium ferrocyanide, and 1 mM MgSO₄ at37° C. for 3 hrs. Viral titers were expressed as LacZ colony formingunits (CFU) per milliliter. CFU was defined in terms of cells stainingblue by X-gal. For SinRep/IL12 vectors, the relative CFU was determinedby RT-PCR with a primer pair specific to Sindbis genomic RNA:5′-AGCTTCCCGCAATTTGAGGT-3′ (SEQ ID NO: 3), 5′-ACGCATGGGGCAGACACAAT-3′(SEQ ID NO: 4). Viral RNA was purified from 300 ml ofOpti-MEM media containing either SinRep/LacZ (control) or SinRep/IL12vector with 1 ml of TRIzol™ reagent (GIBCO BRL). After ethanolprecipitation, all RNA samples were resuspended in 50 ml of RNase-freewater. RT-PCR was performed using the Platinum™ quantitative RT-PCRThermoScrip™ one-step system (GIBCO BRL). Relative CFUs were obtained bycomparing the RT-PCR band intensities of serial dilutions of SinRep/LacZRNA and SinRep/IL12 RNA. The RT-PCR results were then correlated withthe results of X-gal staining assay for SinRep/LacZ. The titers ofSinRep/Luc vectors were assayed on 12-well plates by infection of BHKcells with serial dilutions of 300 ml virus-containing Opti-MEM. Afterovernight incubation, the lucifierase activities in cell lysates fromeach sample were determined using a LUMI-ONE portable luminometer(Bioscan, Inc., Washington, D.C.) for a 30 sec period.

In vitro assays of Sindbis infectivity. To estimate the infectivities ofSindbis virus against hamster and human cells, 300 ml of SinRep/LacZ orSinRep/Luc virus were incubated with 2×10⁵ of BHK, HuH7, LS174T, ES-2,HT29, CFPAC-1, PC-3, HuH7, SKOV-3, A498, HT1197, or A431 cells culturedin 12-well plates at MOI of about 100. The next day, cells were assayedby X-gal staining or the Steady-Glo™ luciferase assay system (PromegaCo.). In the Steady-Glo™ luciferase assay, the cells were aspirated and200 ml of the basal media and 200 ml of the Steady-Glo™ reagent wereadded. Cells were incubated with gentle rocking for 5 min until theybecame detached from the plates. The cell lysates were then transferedto 12×47 mm² cuvettes (PharMingen Co.) and the luciferase activity ofeach was determined using a LUMI-ONE portable luminometer (Bioscan,Inc., Washington, D.C.) for a 30 sec period. For each cell line, 2 to 4independent experiments were performed.

Cell viability assay after Sindbis infection. On day 0, 300 ml of mediumcontaining about 10⁷ SinRep/LacZ vector was added to each of 2×10⁵ BHK,CFPAC-1, ES-2, HT29, LS174T, or SKOV-3 cells cultured in 12-well platesfor one hour. The plates were washed with PBS, refreshed with 1 ml basalmedium and incubated. On the designated day (day 0, 1, 2, 3, 4), cellculture media were collected, and the cells remaining on plates weretrypsinized with 200 ml 1X trypsin EDTA (Mediatech, Inc.). Cellsobtained both from the media and the plates were combined and werecentrifuged for 5 min at 600 rcf (relative centrifugal force). Cellpellets were resuspended in 100 ml PBS and 10 ml of the cell suspensiontransferred to 90 ml trypan blue solution (Mediatech Inc.). 10 ml of thetrypan blue cell mixture were counted by hematocytometry and theviability (%) was determined using formula: clear cells/(clearcells+blue cells)×100%.

Animal models and in vivo transfection. All severe combinedimmunodeficient (SCID) mice (C.B-17-SCID or C.B-17-SCID/bg) used wereobtained from Taconic (Germantown, N.Y.) and were 6-8 weeks old at thetime the experiments were begun. BHK and ES-2 cells were grown assubcutaneous tumors from an initial inoculom of 5′10⁶ cells. Afterapproximately 10 days, the tumors reached the size of at least 1 cm² andtreatment was started and designated as day 1. Tumor-bearing mice weresegregated into three groups: control (n=5), SinRep/LacZ (n=5), andSinRep/IL12 (n=5). 0.5 cc of Opti-MEM containing 10⁷-10⁸ CFU ofSinRep/LacZ vector or SinRep/IL12 vector were injected intraperitoneally(i.p.) into experimental groups. The control group was left untreated orinjected with PBS. The size of tumors was measured daily and calculatedusing the formula: (length, cm)′(width, cm)′(height, cm). SinRep/IL12vector preparations contained high levels (10 ng/ml) mIL12 in theOpti-MEM.

For human tumor models, 4′10⁶ tumor cells such as LS174T, HT29, andCFPAC-1 were grown as subcutaneous tumors for approximately 4 weeksbefore treatment. Tumor-bearing mice were segregated into control (notreatment) and experimental (treated daily with 0.5 cc SinRep/LacZcontaining 10⁷-10⁸ CFU virus) groups. Tumor sizes were recorded dailyusing the formula (length, cm)′(width, cm)′(height, cm). The LS174T testand CFPAC-1 test had 4 mice in both control and experimental group,while HT29 test had 5 mice in both control and experimental group.

Hepatic HuH7 tumors were established by previously described methods(Kozlowski et al., Cancer Res., 1984, 44: 3522-3529). SCID mice (6 weekold; Taconic, Germantown, N.Y.) were anesthetized, and a transverseincision was made in the left flank through the skin and peritoneum,exposing the spleen. Mice were injected with 2×10⁶ HuH7 cells in 250 mlof DMEM containing 10% FBS into the portal vein through the splenichilus using a 27.5-gauge needle (Becton Dickenson). Eight weeks aftertumor injection, when tumor was palpable, mice were injected i.p. with250 ml of Sindbis virus vector once or on three consecutive days.Control mice were injected with Opti-MEM using the same procedure. Inthis experiment, all mice were sacrificed the day after the finalinjection.

b-gal immunostaining. Immunohistochemical detection of β-galactosidaseprotein (β-gal) was performed on formalin-fixed paraffin embeddedtissues using standard streptavidin-biotin horseradish peroxidasecomplex detection with 3,3-diaminobenzidine (DAB) as a chromagen and anautomated immunostainer (NexES, Ventana Medical Systems, Tucson, Ariz.).Appropriate positive (cell line transfected with β-galactosidase vector)and negative controls were used. Briefly, transfected cell lines weregrown under appropriate growth condition to a cell density ofapproximately 10⁶ cells/ml. These cells were gently pelleted andresuspended in a small amount of media. Equal proportions of fibrin andthrombin were added to this suspension to create a fibrin/thrombin/cellclot that was fixed in 10% neutral buffered formalin. Liver/tumorsamples were excised from the animals and fixed in 10% neutral bufferedformalin. Both the cell clot and the tissues were fixed for 12 hrs informalin and processed for paraffin embedding. 5 μm-thick tissuesections were prepared onto electrostatically charged glass slides andbaked at 60° C. overnight. The slides were deparaffinized by threewashes in xylene followed by rehydration through graded alcohols (100%,90%, and 70%). For each sample, one slide was stained with hematoxylinand eosin while the others were used for β-gal detection by staining thecells with a anti-β-galactosidase mouse monoclonal antibody (BIODESIGN,Kennebunk, Me.), which was used at a dilution of 1:50 with overnightincubation. Cellular localization of β-gal protein was cytoplasmic.

b-gal assay. The expression of β-gal protein in tissues was investigatedusing the All-in-One™ b-gal assay reagent kit (PIERCE, Rockford, Ill.).The tissues were homogenized in 3 ml of PBS, with a glass Pyrexhomogenizer using a type B pestle (30 strokes). Homogenized samples werecentrifuged at 1000 rcf at 4° C. for 10 min. The supernatant washarvested, the pellet fraction was resuspended in 2 ml of PBS, andaliquots were harvested as tissue components. 50 ml of each aliquot weremixed with 50 ml of the b-gal assay reagent in a well of a 96-wellplate. After incubation at 37° C. for 30 min, absorbance at 405 nm wasread using a spectrophotometer. Each protein concentration wasdetermined by BIORAD reagent (Bio-Rad Laboratories, Hercules, Calif.)and the results were adjusted per 100 mg of protein.

Statistical analysis of data. The in vitro infectivity data wereanalyzed using a standard student t test. The tumor size data, obtainedfrom different mouse models, were analyzed with repeated-measure two-wayANOVA using GraphPad Prism version 3.0a for Macintosh (GraphPadSoftware, San Diego, Calif.). The significance of each factor ofvariation was determined by the F ratio which is equal to (Mean Squareof the specific factor)/(Residual Mean square). The F ratio is presentin a format: F(df of factor, df of residual) which determines the Fdistribution. P value is the integration of the particular Fdistribution from the F ratio calculated to positive infinity. Forexample, to determine if SinRep/LacZ caused a significant reduction ofBHK tumors, the F ratio for factor SinRep/LacZ was: F(1, 32)=5.915, andthe P value based on the F(1, 32) distribution was 0.0208 which issmaller than 0.05. Therefore the effect of SinRep/LacZ on BHK tumors wasconsidered significant. On the other hand, the effect caused bydifferent individual subjects was not significant since the F (32,32)=0.3712, and the P=0.9968.

The statistical interaction between two factors can be also determinedby the F ratio. If the effects from both SinRep/LacZ and treatment timeare additive, there is no interaction between these two factors. Asdisclosed below, in comparing a tumor size reduction in untreatedanimals with SinRep/LacZ-treated animals, the interaction betweenSinRep/LacZ and treatment time was significant (F(7, 32)=5.14,P=0.0005). In contrast, there was no significant interaction betweenvirus and treatment time, when SinRep/LacZ-treated animals were comparedwith SinRep/IL12-treated animals (F(14, 60)=0.8290, P=0.6303),indicating similar kinetics of tumor regression.

Results

Sindbis vector infects several human tumor cell lines in vitro. Sindbisvirus-based vector SinRep/LacZ (FIG. 1), which carries β-galactosidase(LacZ) gene, and SinRep/Luc, which carries the firefly luciferase (Luc)gene, efficiently infected most human tumor cell lines tested: LS174T(colon), ES-2 (ovarian), HT29 (colon), CFPAC-1 (pancreatic), PC-3(prostate), HuH7 (liver), and SKOV-3 (ovarian). SinRep/Luc also showedlow but significant infectivity of A498 (kidney) and HT1197 (bladder)cells (P<0.0001) and showed intermediate level of infectivity of A431(epidermoid carcinoma) cells (P<0.0001). All human cell lines wereinfected at MOI of about 100 and the marker enzyme (β-gal) activities incell lysates were assayed the next day as described in Materials andMethods section. Mock infections of all human cell lines above resultedin very low relative luminescence unit (RLU) readings of about 150.

Sindbis vector induces cell death upon infection of tumor cells. Sindbisvirus infection of mammalian cells has been previously reported to beextremely cytotoxic due to virally-induced cell apoptosis (Levine etal., Nature, 361:739-742, 1993; Jan and Griffin, J. Virol.,73:10296-10302, 1999; Jan et al., J. Virol., 74:6425-6432, 2000;Balachandran et al., J. Virol., 74: 1513-1523, 2000). To confirm thisobservation for tumor cells, the present inventors tested the viabilityof various tumor cell lines after infection by trypan blue exclusionanalysis. Following infection of six different tumor cell lines withSindbis vector SinRep/LacZ at MOI of about 100, the rapid cell deathoccurred over a five-day period (day 0 to day 4). BHK cells were verysensitive to the apoptosis induced by SinRep/LacZ and the majority ofthem were dead by 2 days after infection. Similar results were observedwith ES-2 cells. LS174T, CFPAC-1, HT29, and SKOV-3 cells required two orthree additional days to achieve the same level of cell death observedwith BHK cells. In control experiments, all cell lines were incubated inmedium only and showed no significant cell death.

Sindbis vector shows anti-tumor effects in vivo. To evaluate theanti-tumor effects of Sindbis vectors, 5×10⁶ BHK cells were inoculatedsubcutaneously in the lower right abdomen of severe combinedimmunodeficient (SCID) mice (8-10 weeks old). The choice of BHK wasbased on their high susceptibility to Sindbis virus infection andSindbis-induced cell death. 10 days after inoculation, BHK tumors hadusually grown to about 1 cm² in size. Mice were then separated intoexperimental and control groups. The experimental group receivedintraperitoneal (i.p.) injections of about 10⁷ SinRep/LacZ vectorscarrying the b-galactosidase reporter gene or SinRep/IL12 vectorscarrying two genes encoding both murine IL12 subunits (mP35 and mP40) tothe left abdomen (site distant to the tumor) five times a week, whilethe control group mice were left untreated or were injected with PBS.All mice in the control group were sacrificed on day 12 of treatmentbecause the tumor burden began to impair the ability to walk as well asother functions. In contrast, mice in the experimental group started toshow tumor reduction on day 6 to 7 of treatment. The repeated-measuretwo-way analysis of variance (RM two-way ANOVA) indicates that Sindbisvectors dramatically reduced the BHK tumor size. In fact, most BHKtumor-bearing mice became tumor-free after 30 days of treatment.Although both SinRep/LacZ and SinRep/IL12 had similar kinetics of theiranti-BHK tumor activity, SinRep/IL12 showed enhanced anti-tumor activityagainst BHK cells compared to SinRep/LacZ (P=0.0167).

Another set of mice were inoculated with 5×10⁶ BHK cells and after 7days developed tumors of about 5×5 mm². Tumor-bearing mice weresegregated into control (no treatment) and experimental (dailySinRep/LacZ treatment) groups, and after three days of consecutivetreatments, slides were prepared from control and experimental tumors.Hematoxylin- and eosin-stained sections revealed two distinct groups oftumors; one group of tumors had approximately 90-95% necrosis while theother group had up to approximately 30% necrosis corresponding totreated and untreated tumors, respectively. The treated tumors weresmaller than the untreated tumors. Vascularity was demonstrated usingimmunohistochemistry for Factor VIII. These blood vessels were medium tosmall sized with the smaller sized blood vessels in the viable tumorareas. Necrotic tumor cells were identified as eosinophilic stainingcells with loss of cellular organization and membranes. The necroticareas in the untreated tumors were located centrally, while, in thetreated tumors, the main mass was necrotic with only a rim of viablecells. Immunohistochemical b-galactosidase (b-gal) staining was obtainedonly in the treated animals and only in the necrotic areas. No b-gal wasdetected in viable tumor areas. In addition, the areas of b-gal-positivestaining corresponded to the areas of Factor VIII-positive staining,demonstrating that Sindbis virus is present and delivered to the tumorby a blood-borne path. Furthermore, the distribution of necrosis andb-gal suggests that viable Sindbis virus is only present at theperiphery of the treated tumors. In the treated tumors, intense andconfined Tunnel-positive signals were observed along the border betweenviable and necrotic areas of the treated tumors. In control tumors, theapoptotic signals on the viable-necrotic border of control tumors wereless intense and more diffused. Also, a larger number of sharp and clearapoptotic bodies were observed in the border regions of treated tumors.No Tunnel signal was observed in the central necrotic area of eithercontrol or treated tumors.

Injections of the SinRep/IL12 vector also resulted in enhanced reductionof BHK tumors in SCID mice (P=0.0167).

Human tumor cells LS174T (colon), HT29 (colon), and CFPAC-1 (pancreatic)were also inoculated subcutaneously and were grown to a defined sizeprior to treatment. SinRep/LacZ treatments were administrated toexperimental groups five times a week and the control groups were leftuntreated or injected with PBS. As in the experiments with BHK tumors,the treatments caused significant LS174T and CFPAC-1 tumor reduction inSCID mice (P<0.0001). After about two weeks of treatment, Sindbis vectorcaused significant growth inhibition in LS174T and CFPAC-1 tumors and anumber of tumor-free mice were also observed in response to treatment.SinRep/LacZ's anti-HT29 tumor activity was lower but still significant(P<0.0001). Based on RM two-way ANOVA analysis, all human tumor modelsshowed no significant effects among different individual subjects.

Sindbis vector is able to target HuH7 tumors in the liver of SCID mice.The HuH7 liver tumors were induced by implantation of humanhepatocellular carcinoma HuH7 cells (2×10⁶) via the portal vein throughthe splenic hilus. After approximately eight weeks, at which time tumorgrowth was evident by palpation, mice were injected i.p. (once or threetimes) with SinRep/LacZ vector. Tumor-bearing mice treated withSinRep/LacZ vector once or for three consecutive days were sacrificedthe day after the final injection. No b-gal positive cells were found innormal liver tissue or in tumors of uninfected control sections. b-galwas also not clearly detectable in the liver tumors which were infectedwith Sindbis vector only once. However, it was easily detectable in theliver tumors which were infected on three consecutive days. In tumorswhich were injected with SinRep/LacZ three times, necrosis was alsoobserved.

In a separate experiment, the expression of b-gal protein was quantifiedin various tissues after one or three treatments. As in the previousexperiment, in tumor cells, there was no significant difference betweenmice subjected to a single infection and control mice, while micereceiving three treatments showed 12- to 18-fold higher b-gal levelsthan control mice in tumor cell supernatants and 19- to 38-fold higherlevels in tissue samples. No significant elevation of b-gal activity wasobserved in liver, heart, lung, kidney, and testis, regardless ofwhether the animals received one or three injections of the Sindbisvector. Low but significant levels of b-gal were observed in the brainsof injected mice. Despite the infectivity to brain cells, all micetreated with one or three SinRep/LacZ injections stayed healthy andshowed no abnormal behavior.

NK cells enhance the anti-tumor effects of Sindbis vectors. SubcutaneousBHK tumors were induced in C.B-17-SCID mice and C.B-17-SCID/bg mice. Thelatter is similar to C.B-17-SCID, except that, in addition to deficiencyof T and B cells, it contains the beige (bg) autosomal recessivemutation, which produces impaired chemotaxis and motility of macrophagesand a deficiency in Natural Killer (NK) cells. Daily treatment withSindbis vectors appeared to be more effective in SCID mice than inSCID/bg (P<0.0001). Thus, complete regression of tumors was obtainedonly in SCID mice.

Discussion

The results disclosed herein indicate that Sindbis virus-basedreplication defective vectors (i.e., SinRep/LacZ, SinRep/Luc, andSinRep/IL12) are capable of infecting a spectrum of human tumor cells invitro and in vivo. As disclosed in the present Example, Sindbis vectorsinduce apoptosis not only in vitro in various mammalian tumor celllines, but also, in vivo in engrafted tumors of human or rodent origin.Thus, even without the addition of any heterologous genes, Sindbisvirus-based vectors can act as therapeutics against malignant tumors.

Indeed, in the present study, entry of Sindbis vectors lead to a highdegree of BHK tumor apoptosis and necrosis in vivo accompanied bycomplete tumor regression. The treatment, which comprised multipleinjections (ranging from three to over fifteen) of Sindbis vectors, didnot appear to be toxic to the experimental animals, indicating thatthese vectors mediate almost selective infection of tumor cells. Indeed,no significant infection of heart, lung, normal liver cells, and kidneywas observed. The minor brain infection, although could be detected,caused no observable clinical CNS disorder in adult (6-8 weeks old)experimental mice, which are known to be resistant to neurovirulenteffects induced by Sindbis virus (Griffin, J. Infect. Dis., 133:456-464, 1976).

As further disclosed herein, the immunostaining of BHK tumors aftertreatments with three injections of SinRep/LacZ demonstrated areas ofextensive necrosis at the periphery of the tumor. This is in contrast tothe typical necrosis seen at the center of tumors caused by hypoxia andpoor nutrition, which can be readily seen in untreated mice.Furthermore, the co-localization of infectious vectors and blood vesselsin the tumor demonstrates that the blood-born characteristic of Sindbisvirus plays an important role in vector delivery.

The vector clearance rate in blood is another important factor, whichdetermines the success of vector-mediated tumor targeting. Both mostwidely used viral vectors, retroviral vectors and adenoviral vectors,have been shown to be unstable in the bloodstream (Miyao et al., Hum.Gene Ther., 8:1575-1583, 1997; Russell et al., Hum. Gene Ther.,6:635-641, 1995; Rother et al., J. Exp. Med., 182:1345-1355, 1995;Alemany et al., J. Gen. Virol., 81:2605-2609, 2000). In contrast,blood-born alphaviruses, including Sindbis virus as well as Sindbisvirus-based vectors, are stable in the bloodstream (Byrnes and Griffin,J. Virol., 74: 644-651, 2000; Bernard et al., Virology, 276: 93-103,2000; Klimstra et al., J. Virol., 72: 7357-7366, 1998).

Despite similarities in in vitro infectivity and cytotoxicity induced bySindbis in various cell lines, differences in anti-tumor efficacy invivo were observed. These differences might result from different numberand the permeability of blood vessels in tumor tissues, from differentlevels of available HALRs, and/or from the presence of additionaltumor-specific receptors for the virus.

As follows from the results of the in vivo experiments disclosed herein,in addition to direct killing of tumor cells induced by Sindbisinfection, the host immune system, in particular, natural killer (NK)cells, which are important for innate immunity, also contribute to thetumor elimination. Indeed, NK cells are well known for theircytotoxicity to tumors (Gumperz and Parham, Nature, 378: 245-248, 1995;Trinchieri, Adv. Immunol., 47: 187-376, 1989) and virus-infected cells(Biron et al., Annu. Rev. Immunol., 17: 189-220, 1999). It has beensuggested that, when the host anti-viral mechanisms become activated,signals are released, such as interferons, which lead to NK cellactivation (Biron et al., 1999, supra). NK cell activation may alsoresult from the inflammation caused by the tumor cell necrosis and therelease of cell contents. IL12 has been shown to be a potent NK cellstimulatory factor, and administration of IL12 has been shown to producepotent anti-tumor and anti-metastatic activity against certain solidtumors (Biron et al., 1999, supra; Brunda et al., J. Exp. Med. 178:1223-1230, 1993; Nastala et al., J. Immunol., 153: 1697-1706, 1994;Takeda et al., J. Immunol., 156: 3366-3373, 1996; Tsung et al., J.Immunol., 158: 3359-3365, 1997). As disclosed herein, the IL12-encodingrecombinant Sindbis vector (SinRep/IL12) possesses enhanced anti-tumorcytotoxicity as compared to the Sindbis vector which does not encodeIL12 (SinRep/LacZ). Since Sindbis vectors of the present inventionexpress exogenous genes at very high levels, additional anti-tumortherapeutic genes such as tumor suppressor, cytotoxic, and cytokinegenes might be ideal candidates to boost the anti-tumor efficacy ofSindbis vectors.

The use of SCID mice (which lack both B and T cells) in the in vivoexperiments described above does not allow to conclusively determinewhether the cytotoxic T cells and neutralizing antibodies would aid ordiminish the anti-tumor activity of the Sindbis vectors of the presentinvention. However, even if these immune cells have a negative effect,this effect can be easily diminished. For example, previously reportedsuccessful sequential vaccinations with alphavirus-based vectors suggestthat the levels of neutralizing antibody elicited after vector injectioncan be reduced by a proper vector design (Kamrud et al., Virology, 263:209-219, 1999; Pushko et al., Virology, 239: 389-401, 1997; Pushko etal., Virology, 239: 389-401, 1997).

Sindbis virus-based vectors demonstrate natural targeting to tumors(probably by taking advantage of a natural differential that exists inthe expression of HALRs on tumors versus normal cells). Beyond thisnatural advantage of Sindbis vectors, targetable Sindbis vectorsrecognizing cell type- or tumor-specific cell surface molecules, whichretain high infectivity and titers, have been developed by the presentinventors (see, e.g., Ohno et al., Nat. Biotechnol., 15: 763-767, 1997),and might allow further refinement of the in vivo targeting. Theabove-presented experimental data suggest that Sindbis vectors, alone orin combination with other existing treatment modalities, are useful as anew tool for cancer gene therapy.

Example 2 Use of Sindbis Vectors to Treat Human Tumors

Replication defective Sindbis virus-based vectors SinRep/LacZ andSinRep/IL12 are prepared as described in Example 1 and administeredintravenously (approximately 500 μl of vector formulation containing10⁷CFU/ml) to patients having advanced (metastatic) melanomas, oradvanced (metastatic) tumors of kidney, brain, colon, prostate, liver,pancreas, bladder, lungs, or ovaries. Treatment is performed 5 times perweek for three or more weeks. The size of tumors is constantly monitoredby MRI and CAT scanning, followed (when possible) by histologicalanalysis of tumor necrosis and biopsy assays for the metastatic behaviorof tumor cells.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying FIGURES. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes,all synthetic concentrations and all molecular weight or molecular massvalues, are approximate, and are provided for description.

All patents, applications, publications, test methods, literature, andother materials cited herein are hereby incorporated by reference.

1. A method for treating a mammal suffering from a tumor that expressesgreater amounts of High Affinity Laminin Receptors (HALRs) than normalcells of the same lineage, which method comprises systemicallyadministering to a mammal harboring such a tumor an amount of areplication defective Sindbis virus vector effective to target and treatthe tumor, wherein the vector has not been modified to target atumor-specific cellular determinant, has a preferential affinity forHALRs, a Sindbis virus E2 HALR binding domain, has genes encodingSindbis proteins nsp1-4 and the vector carries a heterologous anti-tumorgene selected from the group consisting of a suicide gene, anapoptosis-including gene, a tumor suppressor gene, an oncogeneantagonist gene, an immunostimulatory gene, a tumor suppressor effectorgene, an antisense oligonucleotide-encoding sequence, aribozyme-encoding sequence, and an immunogenic peptide-encodingsequence.
 2. The method according to claim 1, wherein the mammal has atleast a partially functional immune system.
 3. The method according toclaim 1, wherein the mammal is a human.
 4. The method according to claim1, wherein the tumor is a solid tumor.
 5. The method according to claim4, wherein the solid tumor is selected from the group consisting of ahepatic carcinoma, melanoma, epidermoid carcinoma, pancreatic cancer,brain malignancy, breast cancer, lung cancer, ovarian adenocarcinoma,colon cancer, prostate cancer, bladder cancer, and renal cancer.
 6. Themethod according to claim 1, wherein said vector is administeredparenterally.
 7. The method of claim 1 wherein said High AffinityLaminin Receptors are unoccupied.
 8. The method according to claim 1,wherein said anti-tumor gene is an apoptosis-inducing gene.
 9. Themethod according to claim 1, wherein the anti-tumor gene is acytokine-encoding gene.
 10. The method of claim 1 wherein said suicidegene is thymidine kinase.
 11. The method of claim 10 comprisingadministering 1-20 mg/day/kg body weight of ganciclovir to said mammal.